KR20210121137A - parathyroid hormone variants - Google Patents

Abstract

The present invention relates to parathyroid hormone (PTH) variants and pharmaceutical compositions comprising the same. The present invention further relates to PTH compositions with improved serum half-life and peak-trough ratio, and methods of controlling serum calcium levels using the PTH variants and compositions of the present invention. The present invention further relates to methods of treating hypoparathyroidism and/or hypocalcemia due to hypoparathyroidism using the PTH variants and compositions of the present invention.

Description

parathyroid hormone variants

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is based on U.S. Provisional Application No. 62/798,113, filed on January 29, 2019, U.S. Provisional Application No. 62/800,744, filed on February 4, 2019, and U.S. Provisional Application No. 62/, filed on August 9, 2019 884,730, the disclosure of which is incorporated herein by reference in its entirety.

technical field

The present invention relates to parathyroid hormone variants and compositions comprising the same. The present invention further relates to parathyroid hormone variants and compositions with improved serum half-life, and methods for controlling serum calcium levels using the parathyroid hormone compositions of the present invention.

Parathyroid hormone (PTH) is a secreted, 84 amino acid product of the parathyroid glands of mammals that controls serum calcium levels by acting on various tissues, including bone. Studies in humans with certain forms of PTH have demonstrated anabolic effects on bone and have generated considerable interest in their use for the treatment of hypoparathyroidism, as well as osteoporosis and related bone disorders.

The first 34 N-terminal amino acids are known to exhibit the same activation as full-length PTH (1-84) at "PTH1R", the only known receptor for PTH. See, eg, Potts et al., Amer. Journ. of Med., 50: 639-649 (1971)]; [Potts et al., Proceed. of the 3rd Intern. Symp. of Endocrin., London, pp 333-349 (1971)]; See Tregear et al., Endocrin., 93: 1349-1353 (1973). Additionally, it has been shown that C-terminal fragments such as 39-84 and 53-84 do not compete with fragments 1-34 for receptor binding. In addition, the C-terminal fragment did not activate adenylate cyclase, all of which led to the conclusion that the C-terminal portion of the PTH peptide is irrelevant. Segre et al., Journ. of Bio. Chem., 254: 6980-6986 (1979)]; [Nissenson et al., Journ. of Bio. Chem., 254: 1469-1475 (1979)]; [Potts et al., Adv. in Prot. Chem., 35: 323-396 (1982).

However, although the C-terminal end of PTH was not considered to be relevant for biological activity, it was found that the C-terminal end of the peptide was required for normal transport and processing. See, eg, Kemper et al., Proceed. of the Nat. Acad. of Sciences, 71: 3731-3735 (1974)]; [Freeman et al., Molec. Endocrin., 1: 628-638 (1987)]; [Wiren et al., Journ. of Bio. Chem., 263: 19771-19777 (1988)]; [Cioffi et al., Journ. of Bio. Chem., 264: 15052-15058 (1989)]; [Karaplis et al., Journ. of Bio. Chem., 270: 1629-1635 (1995)]; See Lim et al., Endocrin., 131: 2325-2330 (1992). In particular, there is evidence that full-length PTH is cleaved into C-terminal fragments within the parathyroid glands, and these fragments are secreted in response to elevated calcium ion concentrations in the blood. There has been no evidence to date that N-terminal fragments other than the intact PTH form are stored in or secreted from the parathyroid gland. Havener et al., Nature-New Biol., 238: 152-154 (1972); [Flueck et al., Journ. of Clin. Invest., 60: 1367-1375 (1977)]; [Mayer et al., Endocrin., 104: 1778-1784 (1979)]; [Chu et al., Endocrin., 93:915-924 (1973)]; [Habener et al., Endocrin., 97: 431-441 (1975)]; [Russell et al., Journ. of Clin. Invest., 72: 1851-1855 (1983)]; [Heinrich et al., Endocrin., 112: 449-458 (1983)]; [Brookman et al., Journ. of Bone & Min. Res., 1: 529-537 (1986)]; [Sherwood et al., Proceed. of the Nat. Acad. of Sciences, 67: 1631-1638 (1970)]; [Arnaud et al., Amer. Journ. of Med., 50: 630-638 (1971)]; [Hanley et al., Journ. of Clin. Invest., 62: 1247-1254 (1978)]; [Di Bella et al., Journ. of Clin. Endocrin. & Metab., 46: 604-612 (1978)]; [Roos et al., Journ. of Clin. Endocrin. & Metab., 53: 709-721 (1981)]; [MacGregor et al., Endocrin., 112: 1019-1025 (1983)]; [Hanley et al., Journ. of Clin. Endocrin. & Metab., 63: 1075-1079 (1986)]; [Morrissey et al., Endocrin., 107: 164-171 (1980)]; [MacGregor et al., Journ. of Biol. Chem., 261: 1929-1934 (1986)]; [MacGregor et al., Journ. of Biol. Chem., 254: 4428-4433 (1979)]; [Kubler et al., Experim. & Clin. Endocrin., 88: 101-108 (1986)]; [Schachter et al., Surgery, 110: 1048-1052 (1991)]; See Tanguay et al., Endocrin., 128: 1863-1868 (1991).

In addition, it is now evident that the C-terminal region of PTH has novel receptors specific for this region of the hormone. See, eg, Hodsman et al., J. Clin. Endocrinol. Metab., 88, pp. 5212-5220 (2003)]. Thus, full-length PTH has different biological properties than that of the N-terminal PTH analog.

However, the importance and effect of full-length parathyroid hormone on bone growth, calcium physiology and replenishment may be, for example, the effect of calcium, but is not so readily understood. The normal daily rise and fall of PTH levels in the blood has a profound effect on bone, and PTH injections can stimulate the growth of new bone in the case of bone loss due to osteoporosis.

Hypoparathyroidism is a lifelong disease characterized by inadequate production of parathyroid hormone (PTH) by the parathyroid gland. Because PTH is important in the regulation of calcium and phosphate levels, loss of PTH decreases blood and bone calcium levels and increases phosphate levels (hypocalcemia and hyperphosphatemia). Hypocalcemia can include, for example, paresthesia, muscle spasms, laryngeal spasms (which can render it incapable of speaking and informing health care providers about the underlying medical condition, which can lead to treatment delays or erroneous treatment), and It possibly causes symptoms such as tetany and neuromuscular hypersensitivity including seizures.

Unlike other successfully formulated proteins, PTH is particularly sensitive to various forms of degradation. For example, oxidation can occur at the methionine residues at positions 8 and 18, resulting in the oxidized PTH species ox-M(8)-PTH and ox-M(18)-PTH, whereas asparagine at position 16 deamidation may occur, thereby generating d16-PTH. Polypeptide chains are truncated by cleavage of peptide bonds at both the N- and C-terminus. Additionally, PTH may also decrease the available concentration of drug by adsorbing to the surface, forming non-specific aggregates, and/or precipitating. All these degradation reactions, and combinations thereof, result in partial or complete loss of PTH bioactivity. Therefore, PTH preparations should prevent this degradation reaction.

Teriparatide (PTH (1-34) sold by Eli Lilly and Co under the trade name Forteo®) has been shown to be an effective alternative to calcitriol calcitriol therapy for hypoparathyroidism. It has been confirmed that normal serum calcium levels can be maintained without hypercalciuria.

Recently, full-length PTH (1-84) was approved as a safe and effective treatment for hypoparathyroidism (sold by Shire-NPS Pharmaceuticals under the trade name NATPARA®). It is the first specific hormone replacement for hypoparathyroidism and is administered subcutaneously once daily and is being taken as a supplement to calcium and vitamin D. However, because of its short half-life in vivo, the human pharmacokinetic profile of natpara exhibits rapid clearance, and high peak-to-trough ratio, resulting in significant intraday serum calcium fluctuations and control of calciumuria, a condition associated with elevated urine calcium levels. cause defects

Accordingly, there is a need for improved PTH receptor agonists, particularly those with long-term activity at the PTH receptor and with a more steady-state physiological profile. Winer KK et al., J. Clin . Endocrinol . Metab , 2003, 88(9), 4214-20.

Various non-limiting aspects and embodiments of the invention are described below.

In one aspect, a parathyroid hormone (PTH) variant is provided. In some embodiments, a PTH variant according to the present invention may be a PTH-Fc fusion protein comprising PTH chemically linked to the Fc region of a human IgG antibody, or a derivative thereof, or a variant thereof. In one embodiment, the PTH linked to an Fc, or variant thereof, may be a full-length PTH (i.e., PTH(1-84)), or a truncated PTH, e.g., C-terminally truncated PTH or a variant thereof. . In one embodiment, the PTH linked to the Fc may be PTH(1-74), or PTH(1-64), or PTH(1-54), or PTH(1-44). In one embodiment, the PTH linked to the Fc is not PTH(1-34). In one embodiment, the PTH linked to the Fc may be full-length PTH (1-84), eg, mutated PTH (1-84).

In one embodiment, the PTH linked to the Fc may be of any length from PTH(1-35) to PTH(1-83). In one embodiment, the PTH linked to the Fc may be PTH(1-33).

In one embodiment, the PTH linked to the Fc is PTH(1-84), or PTH(1-83), or PTH(1-82), or PTH(1-81), or PTH(1-80), or PTH(1-79), or PTH(1-78), or PTH(1-77), or PTH(1-76), or PTH(1-75), or PTH(1-74), or PTH( 1-73), or PTH(1-72), or PTH(1-71), or PTH(1-70), or PTH(1-69), or PTH(1-68), or PTH(1-71) 67), or PTH(1-66), or PTH(1-65), or PTH(1-64), or PTH(1-63), or PTH(1-62), or PTH(1-61) , or PTH(1-60), or PTH(1-59), or PTH(1-58), or PTH(1-57), or PTH(1-56), or PTH(1-55), or PTH(1-54), or PTH(1-53), or PTH(1-52), or PTH(1-51), or PTH(1-50), or PTH(1-49), or PTH( 1-48), or PTH(1-47), or PTH(1-46), or PTH(1-45), or PTH(1-44), or PTH(1-43), or PTH(1- 42), or PTH(1-41), or PTH(1-40), or PTH(1-39), or PTH(1-38), or PTH(1-37), or PTH(1-36) , or PTH(1-35), or PTH(1-33).

In one embodiment, the amino acid sequence of the PTH linked to the Fc comprises a further modification selected from amino acid substitutions, additions, or deletions. In one embodiment, the PTH linked to the Fc comprises a F34A substitution, a F34D substitution, a V35S substitution, or a V35T substitution, or a combination thereof. In one embodiment, the PTH linked to the Fc may be further modified, eg, with modifications such as pegylated, glycosylated, and the like. In one embodiment, the PTH linked to the Fc is PEGylated. In one embodiment, the PTH linked to the Fc is glycosylated. In one embodiment, the Fc is in native Fc form. In another embodiment, the Fc comprises a further modification selected from amino acid substitutions, additions, or deletions. In one embodiment, the Fc is hFcLALA comprising L234A and L235A substitutions. In other embodiments, Fc is administered for increased half-life extension, e.g., as described in Yang et al., mAbs, 2017, 9, 1105, and in other methods commonly known in the art. can be modified accordingly.

In one embodiment there is provided an Fc-linked PTH variant having the amino acid sequence of SEQ ID NOs: 8-11.

In one embodiment there is provided an Fc-linked PTH variant according to the invention as a bivalent construct, ie having two copies of PTH and one copy of Fc.

In another embodiment there is provided an Fc-linked PTH variant according to the invention as a monovalent construct, ie having one copy of PTH and one copy of Fc. In one embodiment, monovalent PTH-Fc variants can be prepared using art-known heavy chain mutations (eg, knob and hole, etc.). One method known in the art is described, for example, in US Pat. No. 8,679,785. In another embodiment, monovalent PTH-Fc variants may be prepared using a monomeric Fc, wherein the two Fc moieties may be linked via a linker, eg, an amino acid linker.

In one embodiment, the serum half-life of a PTH-Fc fusion protein of the invention is greater than that of PTH(1-84). In one embodiment, the PTH-Fc fusion protein of the invention is 2-fold longer, or 3-fold longer, or 5-fold longer, or 10-fold longer, or 20-fold greater than the serum half-life of PTH(1-84). It has a longer, or 30-fold longer, or 40-fold longer serum half-life. In one embodiment, a PTH-Fc fusion protein of the invention has a serum half-life that is 40-fold greater than that of PTH(1-84).

In another embodiment, the peak-to-trough ratio of the PTH-Fc fusion protein is lower than the peak-to-trough ratio of exogenous PTH(1-84). In one embodiment, a PTH-Fc fusion protein of the invention has a peak-to-trough ratio that is at least 2-fold lower than that of PTH(1-84). In one embodiment, a PTH-Fc fusion protein of the invention has a peak-to-trough ratio that is at least 10-fold lower than that of PTH(1-84).

In some embodiments, the PTH variant is processed from a PTH variant precursor polypeptide comprising a signal peptide directly linked to the PTH variant. The signal peptide on the polypeptide can promote secretion of the PTH variant from a mammalian host cell used to produce the PTH variant, and the signal peptide can be cleaved from the PTH variant after secretion. Any number of signal peptides may be used. In one embodiment, the signal peptide may have the following sequence (IgK signal peptide): METPAQLLFLLLLWLPDTTG (SEQ ID NO: 3). In another embodiment, the signal peptide may have the following sequence (IgG heavy chain signal peptide): MEFGLSWLFLVAILKGVQC (SEQ ID NO: 4). In yet another embodiment, the signal peptide may have the following sequence (CD33 signal peptide): MPLLLLLPLLWAGALA (SEQ ID NO: 5). In one particular embodiment, the CD33 signal peptide having the sequence MPLLLLLPLLWAGALA (SEQ ID NO: 5) can provide optimal signal peptide cleavage while leaving position 1 of the PTH variant intact.

In another aspect, there is provided a pharmaceutical composition comprising at least one PTH-Fc fusion protein of the invention and a pharmaceutically acceptable carrier.

In another aspect, a pharmaceutical dosage form comprising at least one PTH-Fc fusion protein of the invention or a pharmaceutical composition of the invention is provided. In one embodiment, the pharmaceutical dosage form of the invention is a liquid dosage form. In one embodiment, the pharmaceutical dosage form is suitable for administration by injection or infusion.

In another aspect, there is provided a nucleic acid encoding a PTH variant according to the present invention. In one embodiment, a nucleic acid encoding a PTH-Fc fusion variant protein is provided.

In another aspect, an expression vector comprising a nucleic acid encoding a PTH variant according to the present invention is provided. In one embodiment, an expression vector comprising a nucleic acid encoding a PTH-Fc fusion variant protein is provided.

In another aspect, there is provided a host cell comprising an expression vector or nucleic acid encoding a PTH variant according to the present invention. In one embodiment, a host cell comprising an expression vector or nucleic acid encoding a PTH-Fc fusion variant protein is provided. In one embodiment, the host cell is a prokaryotic cell, yeast cell, insect cell, or mammalian cell. In one embodiment, the host cell is a CHO cell.

In another aspect, administering to a subject in need of control of serum calcium and phosphate levels a PTH variant of the invention, or a pharmaceutical composition comprising a PTH variant of the invention, or a pharmaceutical dosage form comprising a PTH variant of the invention. A method of controlling serum calcium and phosphate levels in said subject is provided, comprising:

In another aspect, administering to a subject in need of control of urine calcium levels a PTH variant of the invention, or a pharmaceutical composition comprising a PTH variant of the invention, or a pharmaceutical dosage form comprising a PTH variant of the invention. A method of controlling urine calcium levels in the subject is provided, comprising:

In another aspect, comprising administering to a subject having hypoparathyroidism an effective amount of a PTH variant of the invention, or a pharmaceutical composition comprising the PTH variant of the invention, or a pharmaceutical dosage form comprising the PTH variant of the invention. , a method of treating the subject.

In another aspect, comprising administering to a subject having hypoparathyroidism an effective amount of a PTH variant of the invention, or a pharmaceutical composition comprising the PTH variant of the invention, or a pharmaceutical dosage form comprising the PTH variant of the invention. , a method of treating the subject.

In some embodiments, a PTH variant of the invention is administered twice a day, or once a day, or every two days, or every three days, or every 5-8 days. In one embodiment, the PTH variant of the invention is administered once daily. In one embodiment, the PTH variant of the invention is administered once a week. In one embodiment, the PTH variants of the invention are administered subcutaneously.

In some embodiments, the dose of a PTH variant of the invention may need to be titrated to an individual due to within-population variation. In one embodiment, the PTH variants of the invention are from about 1 μg/day to about 500 μg/day, or from about 2 μg/day to about 250 μg/day, or from about 5 μg/day to about 100 μg/day, or about 10 μg/day to about 80 μg/day, or about 20 μg/day to about 100 μg/day, or about 50 μg/day to about 100 μg/day, or about 50 μg/day, or about 60 μg/day day, or about 70 μg/day, or about 80 μg/day, or about 90 μg/day, or about 100 μg/day.

These and other aspects of the invention will become apparent to those skilled in the art after reading the following detailed description of the invention, including the appended claims.

A patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication, including color drawing(s), will be provided by the Patent Office upon request and payment of the necessary fees.
1 is a comparison of serum concentrations of different C-terminally truncated PTH-Fc fusion variants as a function of time after a single subcutaneous administration to rats.
Figure 2(ab) shows serum calcium (Figure 2a ) and as a function of time for PTH-66 (PTH(1-74)-Fc fusion) compared to vehicle (phosphate buffer) control after a single subcutaneous administration in mice Urine calcium ( Figure 2B ) concentrations are presented.
Figure 3 (ab) is provided for PTH-66 (PTH (1-74) -Fc) fusion water PK (Fig. 3a) and PD (FIG. 3b) data for the serum calcium in rats after a single subcutaneous injection TxPTx at various concentrations do.
4(ab) presents data from a single dose PK ( FIG. 4A ) and PD ( FIG. 4B ) study from TPTx rats comparing PTH-66 to PTH(1-84).
5( a d ) presents data from a multi-dose PK ( FIG. 5A ) and PD ( FIGS. 5B-D ) study from daily subcutaneous administration of PTH-66 to TPTx rats.
Figure 6 (ab) proposes a single dose study data PK (Fig. 6a) and PD (Fig. 6b) from the wild type Sino mole Goose monkeys comparing the PTH-66, and PTH (1-84).
7 presents data from a multiple-dose PK and PD study from wild-type cynomolgus monkeys comparing PTH-66 to vehicle after daily subcutaneous dosing.
8A depicts trace chromatographic purification of ˜900 mL CM expressing PTH-66 using a 2× 5 mL Mab Select Sure column. Figure 8b is a flow chart schematically showing the purification of PTH-66 in accordance with embodiments of the invention.
9A depicts the Mab Select crude eluted fraction of PTH-66. Figure 9b shows a Coomassie blue staining of the crude Thank select eluted fractions of PTH-66.
10 depicts SDS-PAGE Coomassie staining of the final PTH-66 product.
11 shows the PTH-66 downstream process flow.
12 is a flow diagram of a production process for PTH-66 that meets a fragmentation target of less than 10%.
13A is a flow chart for the development of a polishing step of a PTH-66 tablet; 13B is a flowchart of a process flow.
14A is a plot of high-throughput screening of Capto MMC ImpRes; 14B is a plot of high-throughput screening of Capto Adhere ImpRes to determine optimal binding conditions; 14C is the chemical structure of Capto MMC; 14D is the chemical structure of Capto Adhere.
15 shows a Capto MMC ImpRes process flow.
16 shows a Capto Adhere ImpRes process flow.
17A is a plot of PTH-66 gradient elution using Capto MMC ImpRes; 17B is a plot of PTH-66 flow-through using Capto Adhere ImpRes.
18A shows a downstream polishing step design for PTH-66; 18B is a graph showing process robustness using stable pools based on impurity clearance and process yield.
19A-F show step yields from pilot plant operation for purification of PTH-66 versus yields from process development for pilot scale production of PTH-66.

Detailed embodiments of the invention are disclosed herein; It should be understood that the disclosed embodiments are merely illustrative of the present invention, which may be embodied in various forms. In addition, each example provided in connection with various embodiments of the present invention is illustrative and not restrictive. Therefore, specific structural and functional details disclosed herein should not be construed as limiting, but only as a representative basis for teaching those skilled in the art to variously utilize the present invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

As used in this specification and the appended claims, the singular forms include plural referents unless the context dictates otherwise. Thus, for example, reference to a “method” includes one or more methods, and/or steps of the type described herein, and/or which will be apparent to one of ordinary skill in the art upon reading this disclosure. will be done

The term "treat" or "treatment" of a condition, disorder or condition refers to (1) a condition, disorder or condition that may be afflicted with, or susceptible to, but is not yet clinical or subclinical of the condition, disorder or condition. preventing, delaying or reducing the likelihood of the onset and/or appearance of at least one clinical or subclinical symptom of a condition, disorder or condition that may occur in a subject not experiencing or exhibiting symptoms; or (2) inhibiting the condition, disorder or condition, i.e., arresting, reducing, or delaying the occurrence of the disease, or its recurrence, or at least one clinical or subclinical symptom thereof; or (3) alleviating the disease, ie, regressing at least one of the condition, disorder or condition, or its clinical or subclinical symptoms. The benefit for the subject to be treated is one that is statistically significant or at least perceptible to the patient or physician.

As used herein, “subject” or “patient” or “individual” or “animal” refers to humans, veterinary animals (eg, cats, dogs, cattle, horses, sheep, pigs, etc.) and laboratory use with disease. animal model (eg, mouse, rat). In a preferred embodiment, the subject is a human.

As used herein, the term “effective” as applied to a dose or amount refers to a quantity of a compound or pharmaceutical composition sufficient to produce the desired activity when administered to a subject in need thereof. It is noted that when a combination of active ingredients is administered, an effective amount of the combination may or may not include amounts of each ingredient that would have been effective if administered separately. The exact amount required will vary from subject to subject depending on the species, age and general condition of the subject, the severity of the condition being treated, the particular drug or drugs used, the mode of administration, and the like.

The phrase “pharmaceutically acceptable,” as used in reference to the compositions of the present invention, refers to molecular entities that are physiologically acceptable and typically do not cause side effects when administered to a mammal (eg, a human) and other of the compositions. refers to the ingredients. Preferably, as used herein, the term "pharmaceutically acceptable" is for use in mammals, and more particularly humans, approved by a regulatory agency of the federal or state government, or in the United States Pharmacopoeia, or listed in other generally accepted pharmacopeias.

As used herein, the terms "carrier" and "diluent" refer to a pharmaceutically acceptable (eg, safe and nontoxic for administration to humans) carrier or diluent material useful in the manufacture of a pharmaceutical formulation. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), pH buffered solution (eg, phosphate buffered saline), sterile saline solution, Ringer's solution or dextrose solution.

As used herein, the terms "fusion protein" and "chimeric protein" refer to a protein resulting from the joining of two or more proteins or portions thereof that are originally distinct. In some embodiments, a linker or spacer will be present between each protein.

As used herein, the term “half-life” is the time it takes for a given quantity, such as a protein concentration or activity, to drop to half of that value measured at the beginning of a period.

As used herein, the terms "ameliorate", "increase" or "reduce", or a grammatical equivalent, refer to a baseline measurement, such as a measurement in the same individual prior to initiating a treatment described herein, or Values compared to measurements in control subjects (or multiple control subjects) in the absence of treatment described herein are shown. A “control subject” is a subject suffering from the same type of disease as the subject being treated and of approximately the same age as the subject being treated.

As used herein, the term "in vitro" refers to an event that occurs in an artificial environment, such as in a test tube or reaction vessel, in cell culture, etc., rather than in a multicellular organism.

As used herein, the term “in vivo” refers to events that occur within multicellular organisms, such as humans and non-human animals. In the context of cell-based systems, the term may be used to refer to events that occur within living cells (as opposed to, for example, in vitro systems).

As used herein, the term "linker" refers to, in a fusion protein, an amino acid sequence or derivative other than that occurring at a particular position in the native protein, and generally to be flexible or between two protein moieties. It is designed to interpose structures such as, for example, α-helices. Linkers are also referred to as spacers. A linker or spacer typically has no biological function per se.

As used herein, the term “polypeptide” refers to a sequential chain of amino acids linked together via peptide bonds. Although the term is used to refer to an amino acid chain of any length, one of ordinary skill in the art would appreciate that the term is not limited to chains that are too long, but will refer to a minimal chain comprising two amino acids linked together via peptide bonds. you will understand that you can As is known to those of ordinary skill in the art, polypeptides can be processed and/or modified. As used herein, the terms "polypeptide" and "peptide" are used interchangeably. The term “polypeptide” may also refer to a protein.

As used herein, the term “PTH variant” includes, without limitation, PTH(1-84) derivatives, including, without limitation, amino acid additions, deletions, and/or substitutions, truncated (eg, C-terminal terminus). truncated) PTH (1-84), PTH fused to a peptide or protein or protein domain either directly, or via a linker or spacer, and PTH post-translationally modified in any manner known in the art (e.g., glycosylated PTH, PEGylated PTH, etc.), or any combination thereof, to any derivative of native PTH (1-84).

PTH

Parathyroid hormone (PTH) is a polypeptide secreted by the parathyroid glands of mammals. Native PTH is 84 amino acids in length and has the following sequence (SEQ ID NO: 1):

As used herein, the term “human PTH” or “hPTH” is encompassed by the term PTH or parathyroid hormone. Human PTH can be synthesized in vivo, recombinantly (in a cell), or synthetically using standard techniques known in the art.

PTH - Fc fusion

In some embodiments, a PTH variant of the invention is a PTH-Fc fusion protein. As used herein, “Fc” refers to the Fc region of a human IgG antibody. Fc-fusion proteins (also known as Fc chimeric fusion proteins, Fc-Ig, Ig-based chimeric fusion proteins and Fc-tag proteins) are Fc domains of IgG chemically linked to a peptide or protein of interest (eg PTH). is composed of

An Fc domain from any IgG can be used. By way of non-limiting example, not only the Fc domains of IgG1, or IgG2, or IgG3, or IgG4, but also various combinations of Fc domains originating from different IgGs, such as, for example, consisting of half IgG2 and half IgG4. can be used In one non-limiting embodiment, the PTH-Fc fusion protein of the invention has the Fc domain of a human IgGl antibody.

Mutations in the Fc region of the antibody may result in silenced effector functions, and different IgGs may have different silencer regions known to those skilled in the art. For example, for IgG1, the following silencer regions are known and described in the art: LALA and N297A (Strohl, W., 2009, Curr. Opin. Biotechnol. vol. 20(6):685- 691); and D265A (Baudino et al., 2008, J. Immunol. 181: 6664-69; Strohl, W., supra). Examples of silent Fc IgG1 antibodies include so-called LALA mutants (“hFcLALA”), which contain L234A and L235A (EU numbering) mutations in the IgG1 Fc amino acid sequence. Another example of a silent IgG1 antibody includes the D265A mutation. Another silent IgG1 antibody contains the N297A mutation, resulting in an aglycosylated/non-glycosylated antibody.

As used herein, IgG1 hFcLALA has the following sequence, wherein the LALA mutation is underlined (SEQ ID NO: 2):

The PTH sequence may be linked directly or indirectly to the Fc domain. In one embodiment, the PTH is linked directly to the Fc domain. In other embodiments, the PTH is linked to the Fc domain by an amino acid linker.

In some embodiments, the Fc region may be further modified to further extend the half-life of the fusion protein. In one non-limiting example, half-life extension techniques, such as NHance technology by ArGenX, can be used in combination with the PTH-Fc variants of the present invention. NHance technology is described, for example, in US Pat. No. 8,163,881, the contents of which are incorporated herein by reference in their entirety.

In one embodiment, the Fc-linked PTH variant according to the invention is provided as a bivalent construct, ie as a construct having two copies of PTH and one copy of Fc.

In another embodiment, the Fc-linked PTH variant according to the invention is provided as a monovalent construct, ie a construct having one copy of PTH and one copy of Fc. In one embodiment, monovalent PTH-Fc variants can be prepared using heavy chain mutations known in the art (eg, knob and hole, etc.). One method known in the art is described, for example, in US Pat. No. 8,679,785. In another embodiment, monovalent PTH-Fc variants may be prepared using a monomeric Fc, wherein the two Fc moieties may be linked via a linker, eg, an amino acid linker.

Signal peptide and protease tags

In some embodiments, a PTH variant of the invention is processed from a PTH variant precursor polypeptide comprising a signal peptide directly linked to the PTH variant. A signal peptide on the polypeptide can promote secretion of the PTH variant from a mammalian host cell used to produce the PTH variant, wherein the signal peptide is cleaved from the PTH variant after secretion. A number of signal peptides may be used.

In some embodiments, an IgK leader sequence having the following sequence (SEQ ID NO: 3) is present in the variants of the invention:

METPAQLLFLLLLWLPDTTG.

In some embodiments, an IgG heavy chain signal peptide having the following sequence (SEQ ID NO: 4) is present in a variant of the invention:

MEFGLSWLFLVAILKGVQC.

In some embodiments, a CD33 signal peptide having the following sequence (SEQ ID NO: 5) is present in a variant of the invention:

MPLLLLLPLLWAGALA.

In one particular embodiment, the CD33 signal peptide having the sequence MPLLLLLPLLWAGALA (SEQ ID NO: 5) can provide optimal signal peptide cleavage while leaving position 1 of the PTH variant intact.

In some embodiments, a poly-histidine tag (HIS tag) is present in the variants of the invention to facilitate PTH protein purification using an affinity chromatography resin that recognizes and binds to the HIS tag. In one embodiment, the TEV-HIS tag is present at the C-terminus of the PTH variants of the invention. The TEV component is a protease recognition site that allows removal of the HIS tag after protein purification. In one embodiment, the sequence of the TEV-HIS tag is ENLYFQSHHHHHH (SEQ ID NO: 6). In one embodiment, ENLYFQ (SEQ ID NO: 7), which is part of the TEV recognition sequence, remains on the protein terminus and does not affect the activity of the protein.

protein glycosylation

As used herein, glycosylation is the reaction in which a carbohydrate (glycan) is attached to a peptide or protein. Different classes of glycans are recognized, the two most prominent being N-linked glycans attached to a nitrogen or arginine side chain and O-linked attached to the hydroxyl oxygen of a serine, threonine, tyrosine, hydroxylysine or hydroxyproline side chain. is a glycan.

In some embodiments, amino acid substitutions to insert glycosylation sites are introduced into the PTH variants of the invention. In some embodiments, such amino acid substitutions may introduce a serine moiety into the PTH variant. In some embodiments, PTH variants of the invention may comprise F34A, F34D, and/or V35S, or V35T mutations to insert a glycan site into the PTH variant.

In some embodiments, an amino acid mutation in F34 (eg, F34A or F34D) and/or V35 (eg, V35S or V35T) occurs at this site during molecular expression in a particular cell type, eg, a CHO cell. This can be done to minimize the observed proteolysis. F34A and F34D mutations reduce proteolysis. The combination of F34A and V35S (or V35T) mutations reduces proteolysis to a much greater extent. This mutation creates a consensus N-linked glycosylation site comprising position N33. This results in glycosylation at position N33 and greatly reduces proteolysis at this position.

In some embodiments, insertion of a glycan site at N33 reduces protein cleavage during host cell expression. Glycans introduce additional size and volume to the PTH variants. Glycan moieties may also improve the solubility of PTH variants. Glycan moieties may also extend the half-life (T1/2) of PTH variants. Glycan insertions at protein mutation sites can also reduce the potential immunogenicity of the mutated sequence. In some embodiments, amino acid substitutions to insert glycosylation sites can be combined with other mutations or sequence truncations or post-translational modifications to PTH variants.

In some embodiments, an amino acid mutation can be made at L59 (eg, L59S or L59T). This mutation creates a glycosylation site at position N57, which can reduce protein cleavage during host cell expression.

Other variants: In some embodiments, amino acid substitutions are introduced into PTH variants to improve developability characteristics. In some embodiments, such amino acid substitutions reduce PTH variant cleavage during expression. In some embodiments, such amino acid substitutions improve the solubility of the PTH variant. In some embodiments, such amino acid substitutions result in glycan insertions. In some embodiments, such amino acid substitutions may introduce aspartate into the sequence.

In some embodiments, the PTH variants of the invention may comprise a F34D mutation to reduce protein cleavage during expression. In some embodiments, PTH variants containing F34D can be combined with other mutations or sequence truncations or post-translational modifications to PTH variants.

Typically, suitable PTH variants are about 2 hours, 3 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 22 hours, 24 hours, 26 hours, has an in vivo half-life of at least 28 hours, 30 hours, 32 hours, 34 hours, 36 hours, 38 hours, 40 hours, 42 hours, 44 hours, 46 hours, or 48 hours. In some embodiments, the PTH variant is 2 to 48 hours, 2 to 44 hours, 2 to 40 hours, 3 to 36 hours, 3 to 32 hours, 3 to 28 hours, 4 to 24 hours, 6 to 24 hours, and 6 It has an in vivo half-life of to 18 hours.

selected parathyroid hormone variant List

In the following list and throughout this specification, the following abbreviations are used:

PTH(1-N) refers to parathyroid hormone peptide residues 1 through N, where N is 84 (for natural-length PTH and variants), or less than 84 (for C-terminal truncated PTH variants) can For example, PTH(1-84) refers to parathyroid hormone peptide residues 1-84 (full length sequence); PTH(1-74) refers to a C-terminal truncated variant of PTH(1-84), which is the first 74 residues of PTH with parathyroid hormone peptide residues 1-74, ie the last 10 residues have been removed; PTH(1-34) refers to a C-terminal truncated variant of the parathyroid hormone peptide residues 1-34, i.e. the first 34 residues PTH(1-84) with the last 50 removed, and so on.

Point mutations in variant sequences are named according to standard convention; For example, F34A means that the phenylalanine at position 34 is replaced by an alanine amino acid; F34D means the phenylalanine at position 34 is replaced by an aspartate residue, V35S means the valine at position 35 is replaced by a serine, and so on.

Δ means [deleted sequence], eg ΔLys means lysine residues have been removed. For the constructs included herein, Lys is removed in the C-terminal region of the Fc molecule, particularly to improve the homogeneity of the drug product.

In one embodiment, the PTH linked to the Fc may be full-length PTH (1-84), eg, mutated PTH (1-84).

In one embodiment, the PTH linked to the Fc may be a truncated PTH of any length from PTH(1-35) to PTH(1-83). In one embodiment, the PTH linked to the Fc may be PTH(1-33).

In some embodiments of the invention, the PTH linked to the Fc is PTH(1-84), or PTH(1-83), or PTH(1-82), or PTH(1-81), or PTH(1-80). ), or PTH(1-79), or PTH(1-78), or PTH(1-77), or PTH(1-76), or PTH(1-75), or PTH(1-74), or PTH(1-73), or PTH(1-72), or PTH(1-71), or PTH(1-70), or PTH(1-69), or PTH(1-68), or PTH (1-67), or PTH(1-66), or PTH(1-65), or PTH(1-64), or PTH(1-63), or PTH(1-62), or PTH(1) -61), or PTH(1-60), or PTH(1-59), or PTH(1-58), or PTH(1-57), or PTH(1-56), or PTH(1-55) ), or PTH(1-54), or PTH(1-53), or PTH(1-52), or PTH(1-51), or PTH(1-50), or PTH(1-49), or PTH(1-48), or PTH(1-47), or PTH(1-46), or PTH(1-45), or PTH(1-44), or PTH(1-43), or PTH (1-42), or PTH(1-41), or PTH(1-40), or PTH(1-39), or PTH(1-38), or PTH(1-37), or PTH(1) -36), or PTH(1-35), or PTH(1-33).

In the following description of exemplary embodiments of the present invention, signal peptides are indicated in italics , PTH regions are indicated in bold , any mutations therein are underlined , and any fusion sequences are indicated by a wavy underline (~~~ ) is indicated.

Molecular name 'PTH-66' (SEQ ID NO: 8)

Description: PTH(1-74)-[F34A, V35S]-hFcLALA-ΔLys

signal peptide , PTH1 -74, mutations that insert a glycan sites, Fc (wavy underline)

Molecular name 'PTH-67' (SEQ ID NO: 9)

Description: PTH(1-64)-[F34A, V35S]-hFcLALA-ΔLys

Signal peptide , PTH1-64 , mutation inserting a glycan site, Fc (wavy underline)

Molecular name 'PTH-68' (SEQ ID NO: 10)

Description: PTH(1-54)-[F34A, V35S]-hFcLALA-ΔLys

Signal peptide, PTH1-54 , mutation inserting a glycan site, Fc (wavy underline)

Molecular name 'PTH-69' (SEQ ID NO: 11)

Description: PTH(1-44)-[F34A, V35S]-hFcLALA-ΔLys

Signal peptide , PTH1-44 , mutation inserting a glycan site, Fc (wavy underline)

Molecular name 'PTH-11' (SEQ ID NO: 12)

Description: PTH(1-84)-hFcLALA

Different PTH variant

In one embodiment, a PTH variant according to the invention may be conjugated to albumin, or a domain of albumin, directly or via a linker. In one embodiment, a PTH-albumin fusion according to the present invention may be prepared by the method described in US Pat. No. 7,592,010. The PTH-albumin variant may be a monovalent construct, ie having one copy of PTH and one copy of albumin, or a domain of albumin. In one embodiment, the serum half-life of the PTH-albumin fusion protein is greater than the serum half-life of PTH(1-84). In one embodiment, the PTH-albumin fusion protein of the invention is 2-fold longer, or 3-fold longer, or 5-fold longer, or 10-fold longer, or 20-fold longer than the serum half-life of PTH(1-84). It has a longer, or 30-fold longer, or 40-fold longer serum half-life. In one embodiment, a PTH-Fc fusion protein of the invention has a serum half-life that is 40-fold greater than that of PTH(1-84).

In one embodiment, the PTH linked to albumin is mutated PTH(1-84), or PTH(1-83), or PTH(1-82), or PTH(1-81), or PTH(1-80) , or PTH(1-79), or PTH(1-78), or PTH(1-77), or PTH(1-76), or PTH(1-75), or PTH(1-74), or PTH(1-73), or PTH(1-72), or PTH(1-71), or PTH(1-70), or PTH(1-69), or PTH(1-68), or PTH( 1-67), or PTH(1-66), or PTH(1-65), or PTH(1-64), or PTH(1-63), or PTH(1-62), or PTH(1- 61), or PTH(1-60), or PTH(1-59), or PTH(1-58), or PTH(1-57), or PTH(1-56), or PTH(1-55) , or PTH(1-54), or PTH(1-53), or PTH(1-52), or PTH(1-51), or PTH(1-50), or PTH(1-49), or PTH(1-48), or PTH(1-47), or PTH(1-46), or PTH(1-45), or PTH(1-44), or PTH(1-43), or PTH( 1-42), or PTH(1-41), or PTH(1-40), or PTH(1-39), or PTH(1-38), or PTH(1-37), or PTH(1- 36), or PTH (1-35), or PTH (1-33).

nucleic acid

In another aspect, a nucleic acid comprising a sequence encoding a PTH variant described herein is provided. The sequence comprises any of SEQ ID NOs: 8-12 and 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 %, 99% or 100% identity. In some embodiments, the nucleic acid may comprise additional non-coding sequences. The nucleic acid may further comprise a designated fragment, variant or consensus sequence thereof, or a deposited vector comprising at least one of these sequences. Nucleic acid molecules may be in the form of RNA, such as mRNA, hnRNA, tRNA, or any other form, or cDNA and genome obtained by cloning, or produced synthetically, for example, via gene therapy using AAV. It may be in the form of DNA, including but not limited to DNA, or any combination thereof. DNA can be triple-stranded, double-stranded or single-stranded, or any combination thereof. Any portion of at least one strand of DNA or RNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the antisense strand.

In some embodiments, a nucleic acid encoding a transgene may be modified to increase expression of the encoded PTH variant, also referred to as codon optimization. For example, a nucleic acid encoding a transgene can be modified by altering the open reading frame for the coding sequence. As used herein, the term "open reading frame" is synonymous with "ORF" and refers to any nucleotide sequence that can potentially encode a protein or portion of a protein. An open reading frame usually begins with a start codon (e.g., for RNA molecules it is denoted as AUG, and for DNA molecules of canonical code it is denoted as ATG), and the frame begins with a stop codon (e.g., for RNA molecules it is denoted as ATG). , UAA, UGA, or UAG, and in DNA molecules of canonical code as TAA, TGA or TAG) are read as codon-triplets. As used herein, the term "codon" refers to a sequence of three nucleotides in a nucleic acid molecule that specifies a particular amino acid during protein synthesis; It is also called triplet or codon-triplet. For example, of the 64 possible codons in the standard genetic code, two codons GAA and GAG encode the amino acid glutamine, while codons AAA and AAG specify the amino acid lysine. In the standard genetic code, three codons are stop codons that do not designate amino acids. As used herein, the term "synonymous codon" means any and all codons that encode a single amino acid. Except for methionine and tryptophan, amino acids are encoded by 2 to 6 synonymous codons. For example, in the standard genetic code the four synonymous codons encoding the amino acid alanine are GCA, GCC, GCG and GCU, the two synonymous codons specifying glutamine are GAA and GAG, and the two synonymous codons encoding lysine are AAA and AAG.

Nucleic acids encoding the open reading frame of PTH variants can be modified using standard codon optimization methods. Various commercial algorithms for codon optimization are available and can be used in practicing the present invention. Typically, codon optimization does not alter the encoded amino acid sequence.

Nucleotide changes may alter synonymous codons within the open reading frame to match endogenous codon usage found in the particular heterologous cell selected to express the PTH variant. Alternatively, or in addition, the nucleotide change may alter the G+C content in the open reading frame to better match the average G+C content of the open reading frame found in the endogenous nucleic acid sequence present in the heterologous host cell. Nucleotide changes may also alter internal regulatory or structural regions or polymononucleotide regions found within the PTH variant sequence. Accordingly, various modifications or optimized nucleotide sequences are envisioned, including, without limitation, nucleic acid sequences that increase expression of PTH variants in prokaryotic cells, yeast cells, insect cells and mammalian cells.

As specified herein, polynucleotides include additional sequences, such as transcribed non-translated sequences that play a role in transcription, mRNA processing (e.g., ribosome binding and stability of mRNA), including splicing and polyadenylation signals. The above-mentioned additional coding sequences, such as at least one signal leader, with or without at least one intron, together with additional non-coding sequences, including but not limited to, non-coding 5' and 3' sequences such as the coding sequence of the fusion peptide; It may further comprise additional coding sequences that encode additional amino acids, such as those that provide additional functionality. Thus, a sequence encoding a PTH variant or designated moiety can be fused to a marker sequence, such as a sequence encoding a peptide that facilitates purification of a fused PTH variant or designated moiety comprising a PTH variant fragment or moiety.

The nucleic acid may further comprise a sequence other than the polynucleotide of the present invention. For example, multiple cloning sites comprising one or more endonuclease restriction sites can be inserted into the nucleic acid to aid in isolation of the polynucleotide. In addition, translatable sequences can be inserted to aid in the isolation of the translated polynucleotides of the invention. For example, the hexa-histidine marker sequence (SEQ ID NO: 13) provides a convenient means of purifying the proteins of the invention. Nucleic acids of the invention, excluding coding sequences, are optionally vectors, adapters or linkers for cloning and/or expression of polynucleotides of the invention.

The coding region of the transgene may contain one or more silent mutations to optimize codon usage for a particular cell type. For example, the codons of a PTH variant can be optimized for expression in vertebrate cells. In some embodiments, the codons of the PTH variants may be optimized for expression in mammalian cells. In some embodiments, the codons of the PTH variants may be optimized for expression in human cells. In some embodiments, the codons of the PTH variants may be optimized for expression in CHO cells.

A nucleic acid sequence encoding a PTH variant as described herein may be molecularly cloned (inserted) into a vector suitable for propagation or expression in a host cell, or in some embodiments an entire plasmid may also be synthesized. . For example, a PTH variant sequence comprising a signal peptide effective to secrete a PTH variant from a host cell is inserted into a suitable vector, such as a sequence selected from SEQ ID NOs: 8-12. Without limitation, prokaryotic expression vectors; yeast expression vectors; A wide variety of expression vectors can be used in practicing the present invention, including, but not limited to, insect expression vectors and mammalian expression vectors. Exemplary vectors suitable for the present invention include, but are not limited to, viral based vectors (eg, AAV based vectors, retroviral based vectors, plasmid based vectors). In some embodiments, a nucleic acid sequence encoding a PTH variant may be inserted into a suitable vector. Typically, a nucleic acid encoding a PTH variant is operably linked to various regulatory sequences or elements.

Various regulatory sequences or elements may be introduced into expression vectors suitable for the present invention. Exemplary regulatory sequences or elements include, but are not limited to, promoters, enhancers, repressors or repressors, 5' untranslated (or non-coding) sequences, introns, 3' untranslated (or non-coding) sequences. .

As used herein, a “promoter” or “promoter sequence” is capable of binding (eg, directly or via other promoter-linked proteins or substances) with an RNA polymerase in a cell and directs transcription of the coding sequence. It is a DNA regulatory region capable of initiation. A promoter sequence is generally joined to its 3' end by a transcription initiation site and extends upstream (5' direction) to contain the minimum number of bases or elements necessary to initiate transcription at any level. A promoter may be operably associated with, or operably linked to, expression control sequences, including enhancer and repressor sequences, or a nucleic acid to be expressed. In some embodiments, the promoter may be inducible. In some embodiments, the inducible promoter may be unidirectional or bidirectional. In some embodiments, the promoter may be a constitutive promoter. In some embodiments, the promoter may be a hybrid promoter, wherein the sequence containing the transcriptional regulatory region is obtained from one source and the sequence containing the transcriptional initiation region is obtained from a second source. Systems for linking control elements to coding sequences within a transgene are well known in the art (General molecular biology and recombinant DNA techniques are described in Sambrook, Fritsch, and Maniatis, Molecular Cloning: A Laboratory Manual , Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989, which is incorporated herein by reference). Commercial vectors suitable for inserting transgenes for expression in a variety of host cells under various growth and induction conditions are also well known in the art.

In some embodiments, a specific promoter for controlling expression of a transgene in a mammalian host cell, such as, but not limited to, an SRa-promoter (Takebe et al., Molec. and Cell. Bio. 8:466-472). (1988)), human CMV very early promoter (Boshart et al., Cell 41:521-530 (1985); Foecking et al., Gene 45:101-105 (1986)), human CMV promoter, Transcription of either human CMV5 promoter, murine CMV very early promoter, EF1-α-promoter, hybrid CMV promoter for liver-specific expression (eg, human α-1-antitrypsin (HAT) or albumin (HAL) promoter) prepared by conjugating a promoter element with the CMV very early promoter), or a promoter for liver cancer specific expression (eg, wherein human albumin (HAL; about 1,000 bp) or human α-1-antitrypsin (HAT, about 2,000 bp) ) in combination with the 145-length enhancer element of human α-1-microglobulin and vicunin precursor gene (AMBP); HAL-AMBP and HAT-AMBP); SV40 early promoter region (Benoist et al., Nature 290:304-310 (1981)), Orgyia pseudotsugata ) very early promoter, herpes thymidine kinase promoter (Wagner et al., Proc. Natl. Acad. Sci. USA 78:1441-1445 (1981)); Alternatively, the regulatory sequence of the metallothionein gene (Brinster et al., Nature 296:39-42 (1982)) may be used. In some embodiments, the mammalian promoter is a constitutive promoter, such as, but not limited to, the hypoxanthine phosphoribosyl transferase (HPTR) promoter, adenosine deaminase promoter, pyruvate kinase promoter, beta-actin promoter, as well as rather, other constitutive promoters known to those of ordinary skill in the art.

In some embodiments, a specific promoter for controlling expression of a transgene in a prokaryotic host cell, such as, but not limited to, the β-lactamase promoter (Villa-Komaroff et al., Proc. Natl. Acad. Sci. USA 75 :3727-3731 (1978)); tac promoter (DeBoer et al., Proc. Natl. Acad. Sci. USA 80:21-25 (1983)); T7 promoter, T3 promoter, M13 promoter, or M16 promoter; In yeast host cells, such as, but not limited to, GAL1, GAL4 or GAL10 promoter, ADH (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter, glyceraldehyde-3-phosphate dehydrogenase Genase III (TDH3) promoter, glyceraldehyde-3-phosphate dehydrogenase II (TDH2) promoter, glyceraldehyde-3-phosphate dehydrogenase I (TDH1) promoter, pyruvate kinase (PYK), in Nolanase (ENO), or triose phosphate isomerase (TPI) may be used.

In some embodiments, the promoter may be a viral promoter, many of which may regulate expression of a transgene in several host cell types, including mammalian cells. Viral promoters that have been shown to drive constitutive expression of coding sequences in eukaryotic cells include, for example, simian virus promoter, herpes simplex virus promoter, papillomavirus promoter, adenovirus promoter, human immunodeficiency virus (HIV) promoter, Rous sarcoma virus promoter, cytomegalovirus (CMV) promoter, long terminal repeat (LTR) of Moloney murine leukemia virus and other retroviruses, thymidine kinase promoter of herpes simplex virus, as well as those of ordinary skill in the art. Other known viral promoters are included.

In some embodiments, the genetic control elements of the expression vector are also 5' non-transcribed and 5' non-translated sequences associated with transcription and translation initiation, respectively, such as TATA box, capping sequence, CAAT sequence, Kozak sequence, etc. may include. Enhancer elements may optionally be used to increase the expression level of an expressed polypeptide or protein. Examples of enhancer elements that have been suggested to act in mammalian cells include the SV40 early gene enhancer as described by Dijkema et al., EMBO J. (1985) 4:761, and Gorman et al., Proc . Natl. Acad. Sci. USA (1982b) 79:6777) and the long terminal repeat (LTR) of human cytomegalovirus as described in Boshart et al., Cell (1985) 41:521. Including enhancers / promoters derived from. The genetic control elements of the expression vector also contain 3' non-transcribed and 3' non-translated sequences associated with transcription and translation termination, respectively, such as polyadenylation (polyadenylation) for stabilization and processing of the 3' end of mRNA transcribed from a promoter. A) It will contain a signal. Exemplary polyA signals include, for example, rabbit beta globin polyA signal, bovine growth hormone polyA signal, chicken beta globin terminator/polyA signal, and SV40 late polyA region.

In some embodiments, the expression vector will preferably but optionally comprise at least one selectable marker. In some embodiments, the selectable marker comprises a resistance gene operably linked to one or more genetic regulatory elements that confers on a host cell the ability to maintain viability when grown in the presence of a cytotoxic chemical and/or drug. It is a nucleic acid sequence that encodes. In some embodiments, a selectable agent may be used to maintain retention of the expression vector in the host cell. In some embodiments, a selectable agent can be used to prevent modification (ie, methylation) and/or silencing of a transgene sequence in an expression vector. In some embodiments, a selectable agent is used to maintain episomal expression of the vector in a host cell. In some embodiments, the selectable agent is used to promote stable integration of a transgene sequence into the host cell genome. In some embodiments, the agonist and/or resistance gene is, in the case of a eukaryotic host cell, methotrexate (MTX), dihydrofolate reductase (DHFR, US Pat. Nos. 4,399,216; 4,634,665; 4,656,134; 4,956,288; 5,149,636; 5,179,017), ampicillin, neomycin (G418), zeomycin, mycophenolic acid, or glutamine synthetase (GS, US Pat. Nos. 5,122,464; 5,770,359; 5,827,739); for prokaryotic host cells, tetracycline, ampicillin, kanamycin or chloramphenicol; and for yeast host cells, URA3, LEU2, HIS3, LYS2, HIS4, ADE8, CUP1 or TRP1.

The expression vector may be transfected, transformed or transduced into a host cell. As used herein, the terms “transfection”, “transformation” and “transduction” all refer to the introduction of an exogenous nucleic acid sequence into a host cell. In some embodiments, an expression vector containing a nucleic acid sequence encoding a PTH Fc fusion is simultaneously transfected, transformed or transduced into a host cell. In some embodiments, an expression vector containing a nucleic acid sequence encoding a PTH variant is sequentially transfected, transformed, or transduced into a host cell.

Examples of transformation, transfection and transduction methods well known in the art include liposomal delivery, i.e., Lipofectamine™ (Gibco) of Hawley-Nelson, Focus 15:73 (1193). ) BRL) method, electroporation, _{CaPO 4} of Graham and van der Erb, Virology, 52:456-457 (1978) Delivery methods, DEAE-dextran mediated delivery, microinjection, biolistic particle delivery, polybrene mediated delivery, cationic mediated lipid delivery, transduction, and viral infections such as, for example, retroviruses, lentiviruses, adenoviruses viruses, adeno-associated viruses and baculoviruses (insect cells).

Once introduced inside the cell, the expression vector can be stably integrated into the genome or exist as an extrachromosomal construct. The vector may also be amplified and multiple copies may be present in the genome or integrated. In some embodiments, a cell of the invention may contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more copies of a nucleic acid encoding a PTH variant. have.

host cell

In another aspect, there is provided a host cell comprising a polynucleotide described herein, eg, permitting expression of a PTH variant in the host cell. The host cell can be a mammalian cell, non-limiting examples include: BALB/c mouse myeloma cell line (NSO/l, ECACC number: 85110503); human retinoblasts [PER.C6, CruCell (Leiden, Netherlands)]; monkey kidney CV1 cell line transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell lines (HEK293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36:59, 1977); human fibrosarcoma cell line (eg, HT1080); young hamster kidney cells (BHK21, ATCC CCL 10); CHO EBNA (Daramola O. et al., Biotechnol. Prog., 2014, 30(1):132-41) and CHO GS (Fan L. et al., Biotechnol. Bioeng. 2012, 109(4):1007- 15), including Chinese hamster ovary cells (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216, 1980); mouse Sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251, 1980); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical carcinoma cells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci., 383:44-68, 1982); MRC 5 cells; FS4 cells; and human liver cancer cell line (Hep G2). In one embodiment, the host cell may be a Chinese hamster ovary cell.

The polynucleotide may be in an expression plasmid. The expression plasmid may have any number of origins of replication known to those of ordinary skill in the art. A polynucleotide or expression plasmid can be introduced into a host cell by a number of ways known to those of ordinary skill in the art. For example, a polynucleotide or expression plasmid can be introduced into a host cell using a flow electroporation system such as a MaxCyte GT®, MaxCyte VLX® or MaxCyte STX® transfection system.

In various embodiments, the host cell expresses a nucleic acid. The host cells can express the PTH variant at levels sufficient for fed-batch cell culture scale or other large scale. Alternative methods of producing recombinant PTH variants on a large scale include roller bottle culture, bioreactor batch culture, and perfusion methods. In some embodiments, the recombinant PTH variant protein is produced by cells cultured in suspension. In some embodiments, the recombinant PTH variant protein is produced by an adherent cell.

Produce

Recombinant PTH variants may be produced by any available means. For example, recombinant PTH variants can be produced recombinantly by using host cell systems engineered to express recombinant PTH variant-encoding nucleic acids. Alternatively, or additionally, recombinant PTH variants can be produced by activating an endogenous gene. Alternatively, or additionally, recombinant PTH variants can be prepared partially or fully by chemical synthesis. Alternatively, recombinant PTH variants can be produced in vivo by mRNA therapeutics or AAV/lentiviral gene therapy.

In some embodiments, in some embodiments, the recombinant PTH variant is produced in a mammalian cell. Non-limiting examples of mammalian cells that can be used in accordance with the present invention include, but are not limited to, the BALB/c mouse myeloma cell line (NSO/1, ECACC number: 85110503); human retinoblasts [PER.C6, Krussel: Leiden, Netherlands); monkey kidney CV1 cell line transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell lines (HEK293 or 293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36:59, 1977); human fibrosarcoma cell line (eg, HT1080); young hamster kidney cells (BHK21, ATCC CCL 10); CHO EBNA (Daramola O. et al., Biotechnol. Prog., 2014, 30(1):132-41) and CHO GS (Fan L. et al., Biotechnol. Bioeng. 2012, 109(4):1007- 15), including Chinese hamster ovary cells +/-DHFR (CHO, Urlaub and Chasin, Proc. Natl. Acad. Sci. USA, 77:4216, 1980); mouse Sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251, 1980); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1 587); human cervical carcinoma cells (HeLa, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci., 383:44-68, 1982); MRC 5 cells; FS4 cells; and human liver cancer cell line (Hep G2).

In some embodiments, the recombinant PTH variant is produced from a human cell. In some embodiments, the recombinant PTH variant is produced from CHO cells or HT1080 cells.

In certain embodiments, host cells are selected to generate a cell line based on certain desirable attributes or growth under certain conditions selected for cell culture. Those skilled in the art will recognize that such attributes can be ascertained based on known characteristics and/or traits of an established cell line (ie, a characterized commercially available cell line) or through empirical evaluation. . In some embodiments, cell lines may be selected for their ability to grow in a feeder layer of cells. In some embodiments, cell lines may be selected for their ability to grow in suspension. In some embodiments, cell lines may be selected for their ability to grow as adherent monolayers of cells. In some embodiments, a cell line can be selected for preferential post-translational modification (eg, glycosylation. In some embodiments, in some embodiments, such cells are any tissue culture vessel or any treated with a suitable adhesion substrate). In some embodiments, a suitable adhesion matrix is collagen (eg, collagen I, II, II or IV), gelatin, fibronectin, laminin, vitronectin, fibrinogen, BD Matrigel (BD Matrigel). )™, basement membrane matrix, dermatan sulfate proteoglycan, poly-D-lysine and/or combinations thereof In some embodiments, adherent host cells are selected under specific growth conditions to grow in suspension and This method of transforming adherent cells and growing them in suspension is known in the art.For example, the cells can be grown in suspension culture by gradually removing animal serum from the growth medium over time. It can be regulated to grow.

Typically, a cell engineered to express a recombinant PTH variant may comprise a transgene encoding a recombinant PTH variant described herein. It should be appreciated that the nucleic acid encoding the recombinant PTH variant may contain regulatory sequences, genetic control sequences, promoters, non-coding sequences and/or other suitable sequences for expressing the recombinant PTH variant. Typically, a coding region is operably linked with one or more of these nucleic acid components.

In some embodiments, the PTH variant is expressed using a batch culture method. In some embodiments, the batch culture period may last for 7-14 days. In some embodiments, batch culture may proceed for 14-21 days. In some embodiments, the PTH variant is expressed using a perfusion culture method (collecting culture medium over time daily). In some embodiments, the PTH variant is expressed using a pseudoperfusion culture method (collecting culture medium at a single time point daily with replacement of fresh medium). In some embodiments, specific feeding regimens/media may be used to improve optimal PTH variant production (improving glycans, reducing clipping). In some embodiments, cell density can be controlled/maintained to improve optimal PTH variant production (improving glycans/reducing clipping).

In some embodiments, the recombinant PTH variant is produced in vivo by an mRNA therapeutic agent. mRNA encoding the PTH variant is prepared and administered to a patient in need of the PTH variant. The mRNA may comprise a sequence corresponding to the DNA sequence of SEQ ID NOs: 8-12. Various routes of administration can be used, such as injection, nebulization in the lungs, and electroporation under the skin. mRNA may be encapsulated in a viral vector or a non-viral vector. Exemplary non-viral vectors include liposomes, cationic polymers, and cubosomes.

Recovery and purification

A variety of means can be used to purify PTH variants from cells. A variety of methods can be used to purify or isolate PTH variants produced according to the various methods described herein. In some embodiments, the expressed protein is secreted into the medium, so that cells and other solids can be removed, for example, by centrifugation or filtration as a first step in the purification process. Alternatively, or additionally, the expressed protein is associated with the surface of the host cell. In such embodiments, host cells expressing the polypeptide or protein are lysed for purification. Lysis of mammalian host cells can be accomplished by a number of means well known to those skilled in the art, including physical disruption by glass beads, the use of surfactants, and exposure to high pH conditions. In some embodiments, the PTH variant may be expressed as an insoluble fraction (inclusion body). In such embodiments, cells will be harvested, for example, by centrifugation and lysed using denaturants commonly known in the art.

PTH variants can be prepared by standard methods including, but not limited to, chromatography (e.g., ion exchange, affinity, size exclusion and hydroxyapatite chromatography), gel filtration, centrifugation or differential solubility, ethanol precipitation, or by any other available technique for the purification of proteins. See, eg, Scopes, Protein Purification Principles and Practice 2nd Edition, Springer-Verlag, New York, 1987; [Higgins, S. J. and Hames, B. D. (eds.), Protein Expression: A Practical Approach, Oxford Univ Press, 1999]; and Deutscher, MP, Simon, MI, Abelson, JN (eds.), Guide to Protein Purification: Methods in Enzymology (Methods in Enzymology Series, Vol 182), Academic Press, 1997, all of which are incorporated herein by reference. included). In particular, in the case of immunoaffinity chromatography, the protein can be isolated by binding to an affinity column comprising an antibody generated against the protein and attached to an immobilized support. Alternatively, affinity tags such as influenza coat sequence, poly-histidine or glutathione-S-transferase can be attached to the protein by standard recombinant techniques for easy purification by passage through an appropriate affinity column. A protease inhibitor such as phenyl methyl sulfonyl fluoride (PMSF), leupeptin, pepstatin or aprotinin may be added at any or all stages to reduce or eliminate degradation of the polypeptide or protein during the purification process. Protease inhibitors are particularly preferred when cells must be lysed to isolate and purify the expressed polypeptide or protein.

PTH variants or designated moieties can be purified by protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, mixed mode chromatography (e.g. For example, it can be recovered and purified from recombinant cell culture by well-known methods including, but not limited to, MEP Hypercel™), hydroxylapatite chromatography, and lectin chromatography. High performance liquid chromatography (“HPLC”) can also be used for purification. See, e.g., Colligan, Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, N.Y. (1997-2003)].

Neutralization should be performed carefully after protein A purification to avoid aggregation/precipitation of the PTH-Fc fusion protein.

The PTH variants or designated portions of the present invention may contain products that are naturally purified, products of chemical synthesis methods, and products produced by recombinant techniques from eukaryotic hosts, including, for example, yeast, higher plant, insect and mammalian cells. include Depending on the host and particular PTH variant employed in the recombinant production method, the PTH variant or designated portion of the invention may be glycosylated or non-glycosylated.

formulation

In some embodiments, the pharmaceutical compositions described herein further comprise a carrier. Suitable acceptable carriers are water, salt solutions (eg NaCl), saline, buffered saline, alcohol, glycerol, ethanol, gum arabic, vegetable oil, benzyl alcohol, polyethylene glycol, gelatin, carbohydrates such as lactose, amylose. or starches, sugars such as mannitol, sucrose and the like, dextrose, magnesium stearate, talc, silicic acid, viscous paraffin, perfume oils, fatty acid esters, hydroxymethylcellulose, polyvinyl pyrrolidone, and the like, as well as combinations thereof. It is not limited thereto. Pharmaceutical preparations may, if desired, contain adjuvants which do not detrimentally react with or interfere with the activity of the active compound (e.g., diluents, buffers, lipophilic solvents, preservatives, adjuvants, lubricants, preservatives, stabilizers, wetting agents, emulsifiers) , salts, buffers, coloring agents, flavoring agents and/or aromatic substances to affect osmotic pressure). In some embodiments, an aqueous carrier suitable for intravenous administration is used.

Pharmaceutically acceptable adjuvants are preferred. The sterilization ratio of the solution-limiting examples and the production method is, for example, but are not limited to, the literature [Gennaro, Ed, Remington's Pharmaceutical Sciences, 18 ^th Edition, Mack Publishing Co. (Easton, Pa.) 1990], is well known in the art. A pharmaceutically acceptable carrier suitable for the mode of administration, solubility and/or stability of the PTH variant composition as well known in the art or as described herein can be conveniently selected. For example, sterile saline and phosphate buffered saline at slightly acidic or physiological pH can be used. pH buffering agents include phosphate, citrate, acetate, tris(hydroxymethyl)aminomethane (TRIS), N-tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPS), ammonium bicarbonate, diethanolamine, histidine (preferred buffers), arginine, lysine or acetate or mixtures thereof. Preferred buffer ranges are pH 4-8, or pH 6.5-8, or pH 7-7.5. Preservatives such as para, meta and ortho-cresol, methyl- and propyl-parabens, phenol, benzyl alcohol, sodium benzoate, benzoic acid, benzyl-benzoate, sorbic acid, propanoic acid, esters of p-hydroxybenzoic acid are included in the pharmaceutical composition. may be provided. Stabilizers that prevent oxidation, deamidation, isomerization, racemization, cyclization, peptide hydrolysis, such as, for example, ascorbic acid, methionine, tryptophan, EDTA, asparagine, lysine, arginine, glutamine and glycine are pharmaceuticals. may be provided in a composition. Stabilizers to prevent aggregation, fibrillation and settling, such as sodium dodecyl sulfate, polyethylene glycol, carboxymethyl cellulose, cyclodextrin, may be provided in the pharmaceutical composition. Organic modifiers such as ethanol, acetic acid or acetate and salts thereof for solubilization or anti-aggregation may be provided in the pharmaceutical composition. An isotonicity maker such as a salt such as sodium chloride or a carbohydrate such as dextrose, mannitol, lactose, trehalose, sucrose or mixtures thereof may be provided in the pharmaceutical composition.

Pharmaceutical excipients and additives useful in the present compositions include proteins, peptides, amino acids, lipids and carbohydrates (e.g., sugars including monosaccharides, di-, tri-, tetra-, and oligosaccharides; derivatized sugars such as alditol, aldonic acid, esterified sugars, etc.; and polysaccharides or sugar polymers), which may be present alone or in combination, alone or in combination, at 1-99.99% by weight or included in % by volume. Exemplary protein excipients include serum albumin, such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acid/PTH variant components that may also act in buffering capacity include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, etc. . One preferred amino acid is glycine.

carbohydrate excipients such as monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose and the like; disaccharides such as lactose, sucrose, trehalose, cellobiose and the like; polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch and the like; and alditols such as mannitol, xylitol, maltitol, lactitol, xylitol, sorbitol (glucitol), myoinositol and the like can be used.

The PTH variant composition may also include a buffer or pH adjuster; Typically, the buffer is a salt prepared from an organic acid or base. Exemplary buffers include organic acid salts such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid or phthalic acid; tris, tromethamine hydrochloride or phosphate buffers.

Additionally, the PTH variant compositions of the present invention may contain polymeric excipients/additives such as polyvinylpyrrolidone, picol (polymeric sugar), dextrates (eg, cyclodextrin, such as 2-hydroxypropyl-β-cyclodextrin). ), polyethylene glycols, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, surfactants (e.g., polysorbates such as "TWEEN 20" and "Tween 80"), lipids (e.g. , phospholipids, fatty acids), steroids (eg, cholesterol), and chelating agents (eg, EDTA).

These and further known pharmaceutical excipients and/or additives suitable for use in the PTH variant compositions according to the invention are described, for example, in "Remington: The Science & Practice of Pharmacy", 21 ^st. ed., Williams & Williams, (2005)], and ["Physician's Desk Reference", 71 ^st. ed., Medical Economics, Montvale, NJ (2017)], the disclosure of which is incorporated herein by reference in its entirety. Preferred carrier or excipient materials are salts (eg sodium chloride), carbohydrates (eg mannitol) and buffers (eg citrate).

Pharmaceutical compositions may be formulated as liquids suitable for administration by intravenous or subcutaneous injection or infusion. Such liquids may include one or more solvents. Exemplary solvents include water; alcohols such as ethanol and isopropyl alcohol; vegetable oil; polyethylene glycol; propylene glycol; and glycerin or mixtures and combinations thereof. Aqueous carriers suitable for intravenous administration may be used. For example, in some embodiments, compositions for intravenous administration are typically solutions in sterile isotonic aqueous buffer. If desired, the composition may also include a solubilizing agent and a local anesthetic to relieve pain at the injection site. In general, the ingredients are supplied separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or anhydrous concentrate, in a closed container such as an ampoule or sachette indicating the quantity of the active agent. Where the composition is to be administered by infusion, it may be presented as an infusion bottle containing sterile pharmaceutical grade water, saline or dextrose/water. When the composition is administered by injection, an ampoule of sterile water for injection or saline may be provided so that the ingredients can be mixed prior to administration.

As mentioned above, the formulation preferably comprises at least one PTH variant in a pharmaceutically acceptable formulation, as well as optional preserved solutions and formulations containing saline or a suitable buffer comprising a selected salt, as well as a preservative. , a multi-use preserved formulation suitable for pharmaceutical or veterinary use. Preserved preparations may contain at least one known preservative or at least one of phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride (e.g., hexahydrate), alkylparabens (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal, or mixtures thereof in an aqueous diluent containing a preservative optionally selected from the group. Any suitable concentration or mixture as known in the art, such as 0.001-5%, or any range or value therein, such as, but not limited to, 0.001, 0.003, 0.005, 0.009, 0.01, 0.02, 0.03, 0.05, 0.09, 0.1, 0.2, 0.3, 0.4., 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2 , 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.3, 4.5, 4.6, 4.7, 4.8, 4.9, or Any range or value therein may be used. Non-limiting examples include preservative free, 0.1-2% m-cresol (eg, 0.2, 0.3, 0.4, 0.5, 0.9, 1.0%), 0.1-3% benzyl alcohol (eg, 0.5, 0.9) , 1.1., 1.5, 1.9, 2.0, 2.5%), 0.001-0.5% thimerosal (eg 0.005, 0.01), 0.001-2.0% phenol (eg 0.05, 0.25, 0.28, 0.5, 0.9) , 1.0%), 0.0005-1.0% alkylparaben(s) (eg, 0.00075, 0.0009, 0.001, 0.002, 0.005, 0.0075, 0.009, 0.01, 0.02, 0.05, 0.075, 0.09, 0.1, 0.2, 0.3, 0.5 , 0.75, 0.9, 1.0%) and the like.

PTH variants may be formulated for parenteral administration and may contain, as conventional excipients, sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of plant origin, hydrogenated naphthalene, and the like. Aqueous or oleaginous suspensions for injection can be prepared according to known methods by using suitable emulsifying or wetting agents and suspending agents. An injectable agent may be a non-toxic, parenterally administrable diluent such as a sterile injectable solution or suspension in an aqueous solution or solvent. As a usable vehicle or solvent, water, Ringer's solution, isotonic saline, and the like are acceptable; As conventional solvents or suspending solvents, sterile non-volatile oils may be used. For this purpose, natural or synthetic or semi-synthetic fatty oils or fatty acids; Any kind of non-volatile oils and fatty acids may be used, including natural or synthetic or semi-synthetic mono- or di- or tri-glycerides. Parenteral administration is known in the art and includes, but is not limited to, conventional means of injection, gas pressurized needleless injection devices as described in U.S. Patent No. 5,851,198, and laser puncture devices as described in U.S. Patent No. 5,839,446. it doesn't happen

The pharmaceutical composition may be an extended release formulation. The pharmaceutical composition may also be formulated for sustained release, extended release, delayed release or slow release, for example, of a PTH variant comprising the amino acid sequence of SEQ ID NO:8. Extended release, also known as controlled release and sustained release, may be provided in an injectable formulation. Microspheres, nanospheres, implants, depots and polymers can be used in combination with any of the compounds, methods and agents described herein to provide an extended release profile.

PTH variants of the invention, e.g., comprising the amino acid sequence of SEQ ID NO: 8, are from about 0.001 to about 100 mg/mL, or from about 0.005 to about 50 mg/mL, or from about 0.007 to about 20 mg/mL. , or from about 0.01 to about 10 mg/mL, or from about 0.05 to about 5.0 mg/mL, or from about 0.07 to about 2.0 mg/mL, or from about 0.1 to about 1.0 mg/mL. In one embodiment, the PTH variant may be formulated at a concentration of about 0.01 to about 10 mg/mL. In one embodiment, the PTH variant may be formulated at a concentration of about 0.1 to about 1.0 mg/mL.

Formulations and compositions comprising the PTH variant optionally include drugs related to diabetes or insulin metabolism, anti-infective drugs, cardiovascular (CV) drugs, central nervous system (CNS) drugs, autonomic nervous system (ANS) drugs, respiratory drugs, gastrointestinal (GI) drugs ) at least one compound selected from vascular drugs, hormonal drugs, drugs for fluid or electrolyte balance, blood drugs, anti-neoplastic drugs, immunomodulatory drugs, ophthalmic, otic or nasal drugs, topical drugs, nutritional drugs, etc. It may further include a protein in an effective amount. Such drugs are well known in the art, including the formulations, indications, dosing and administration set forth for each herein (see, e.g., Nursing 2001 Handbook of Drugs, 21 ^st. edition, Springhouse Corp., Springhouse, Pa., 2001]; [Health Professional's Drug Guide 2001, ed., Shannon, Wilson, Stang, Prentice-Hall, Inc, Upper Saddle River, NJ]; See Pharmacotherapy Handbook, Wells et al., ed., Appleton & Lange, Stamford, CT (each of which is incorporated herein by reference in its entirety).

The PTH variant may also be formulated as a slow release implantable device for prolonged or sustained administration of the PTH variant. The sustained release formulation may be in the form of a patch placed outside the body. Examples of sustained release formulations include complexes of biocompatible polymers such as poly(lactic acid), poly(lactic acid-co-glycolic acid), methylcellulose, hyaluronic acid, sialic acid, silicates, collagen, liposomes, and the like. Sustained release formulations may be of particular interest when it is desirable to provide high local concentrations of PTH variant peptibodies.

PTH variant compositions and formulations are lyophilized at least one PTH variant (e.g., comprising the amino acid sequence of SEQ ID NO: 8) or a designated portion or reconstituted as a clear solution or into a second vial containing an aqueous diluent It may be provided to the patient as a double vial comprising a vial of the variant. A single solution vial or dual vial requiring reconstitution can be reused multiple times and may be sufficient to treat a patient for one or multiple cycles, thus providing a more convenient treatment regimen than currently available. have.

The PTH variant compositions and formulations may be prepared in pharmacies, clinics or other institutions and establishments such as lyophilized at least one PTH variant (e.g., SEQ ID NO: : 8) or indirectly provided to the patient by providing a double vial containing a vial of the designated portion or variant. In such cases, the clear solution may be up to 1 liter or even larger in size, which provides a large reservoir therefrom for a PTH variant (e.g., comprising the amino acid sequence of SEQ ID NO:8) or a designated portion or variant solution. Smaller portions of these may be withdrawn in one or multiple batches, transferred to smaller vials, and provided to customers and/or patients at a pharmacy or clinic. The article may include packaging material. In addition to the information required by regulatory agencies, the packaging material can provide the conditions under which the product can be used. The packaging material may be provided to the patient by reconstituting the PTH variant (eg, comprising the amino acid sequence of SEQ ID NO: 8) or the designated moiety or variant in an aqueous diluent to form a solution, and dispensing the solution into two vials, wet/dry For a product, we can provide guidance for use over a period of 2-24 hours or longer.

cure

In another aspect, treating the subject with a PTH variant of the invention with a PTH variant of the invention using a dosing regimen effective to control serum and urine calcium levels in a subject having hypoparathyroidism and/or hypocalcemia due to hypoparathyroidism is indicated. Provided is a method of treating a subject having hypoparathyroidism and/or hypocalcemia due to hypoparathyroidism, comprising: Because the PTH variant has a longer half-life than native PTH (1-84), it may be particularly effective in treating hypoparathyroidism and/or hypocalcemia due to hypoparathyroidism. Due to the greater half-life, the PTH variant can be administered and maintained daily at more physiologically appropriate levels, thereby providing more sustained control over both serum and urine calcium.

In some embodiments, the method is effective in controlling serum calcium levels in a subject. In some embodiments, the method comprises increasing the serum calcium level of a subject having hypoparathyroidism and/or hypocalcemia due to hypoparathyroidism as compared to the serum calcium level of the subject in the absence of a PTH variant of the present invention. effective. In one embodiment, the method is effective to maintain a subject's serum calcium level at a lower peak-to-trough ratio as compared to the serum calcium level of a subject receiving exogenous native PTH (1-84). In one embodiment, the method is effective in reducing the amount of calcium supplementation in a subject with hypoparathyroidism.

In some embodiments, the method is effective in controlling urine calcium secretion in a subject. In one embodiment, the method is effective in reducing urine calcium excretion to the level of a healthy volunteer (reducing potential renal complications).

For example, a PTH variant comprising the amino acid sequence of SEQ ID NO:8 may be administered subcutaneously or intravenously. In various embodiments, multiple administrations are performed according to a dosing regimen. As used herein, the term "QD" or "q.d." refers to once daily dosing, "Q2D" refers to dosing every two days, and the like. "QW" means weekly dosing. "BID" means twice daily administration. Dosing can be, for example, BID, once daily (QD), Q2D, Q3D, Q4D, Q5D, Q6D, QW, once every 8 days, once every 9 days, once every 10 days, 1 every 11 days. once every 12 days, once every 13 days, once every 2 weeks, once every 15 days, once every 16 days, or once every 17 days, once every 3 weeks or once a month have.

The PTH variant (eg, comprising the amino acid sequence of SEQ ID NO: 8) is 0.001 twice daily, or once daily, or every 2 days, every 5-8 days, or weekly (QW). It may be administered subcutaneously according to a dosage regimen of μg to 1,000 μg. Alternatively, the PTH variant may be administered, eg, every 3 weeks or once a month, for maintenance purposes.

PTH variants (eg, comprising the amino acid sequence of SEQ ID NO: 8) can be administered once daily (QD) from about 1 μg/day to about 500 μg/day, or from about 2 μg/day to about 250 μg/day. day, or from about 5 μg/day to about 100 μg/day, or from about 10 μg/day to about 80 μg/day, or from about 20 μg/day to about 100 μg/day, or from about 20 μg/day to about 100 μg/day, or about 50 μg, or about 60 μg, or about 70 μg, or about 80 μg, or about 90 μg, or about 100 μg. The PTH variant (eg, comprising the amino acid sequence of SEQ ID NO: 8) may be administered at a concentration of 0.001 to 1,000 μg/mL.

The dosing regimen is 1 month, or 2 months, 6 months, or 1 year, or 2 years, or 5 years, or more than 5 years for treating hypoparathyroidism and/or hypocalcemia due to hypoparathyroidism. can be performed over a period of time. The PTH variant may be administered for the lifetime of the subject for maintenance.

As used herein, the term “subcutaneous tissue” is defined as the layer of loose, irregular connective tissue that lies just below the skin. For example, subcutaneous administration can be performed by injecting the composition into a site including, but not limited to, the femoral region, the abdominal region, the gluteal region, or the scapular region. For this purpose, the formulation may be injected using a syringe. However, other devices for administration of agents, such as injection devices (eg, Inject-ease™ and Genject™ devices); syringe pens (such as Q-Cliq™ and GenPen™); needleless devices (eg, MediJector™ and BioJector™); and subcutaneous patch delivery systems are available. In some embodiments, for example, a PTH variant comprising the amino acid sequence of SEQ ID NO:8, or a pharmaceutical composition comprising the same, is administered intravenously.

In various embodiments, said method of treating hypoparathyroidism and/or hypocalcemia due to hypoparathyroidism is used in combination with other methods of treating hypoparathyroidism and/or hypocalcemia due to hypoparathyroidism. .

Example

The following examples illustrate specific aspects of the present description. These examples merely provide specific understanding and practice of the embodiments and various aspects thereof, and these examples should not be construed as limiting.

Example 1: Fc fusion PTH variant (exemplified by PTH-66) production and purification

PTH-66 was transiently expressed in CHO cells and the conditioned medium was stored at -20°C until further use. Conditioned medium was thawed in a 25° C. water bath and filtered through a 0.2 μm bottle top filter device. The PTH-66 protein was purified according to the manufacturer's protocol on Protein A-derived resin MabSelect Sure ( FIG. 8 ). The MabSelect Sure column was pre-equilibrated with PBS. Thawed conditioned medium was loaded onto the column and then washed thoroughly with PBS. PTH-66 protein was eluted by step elution with 100 mM sodium citrate buffer, pH 3.2. Elution fractions were analyzed on SDS-PAGE ( FIG. 9 ). The pH of the pooled elution fractions was brought to about pH 6 using 1 M Tris (pH 10). The neutralized pooled elution fractions were dialyzed against PTH-66 storage buffer 20 mM sodium phosphate buffer, 50 mM sodium chloride, 26 mg/ml mannitol, pH 6.0 and stored at -80°C.

8A depicts trace chromatographic purification of ˜900 mL CM expressing PTH-66 using a 2× 5 mL Mab Select Sure column. Figure 8b is a flow chart schematically showing the purification of PTH-66 in accordance with embodiments of the invention.

9A depicts the Mab Select crude eluted fraction of PTH-66. 9B depicts Coomassie blue staining for the Mab Select crude eluted fraction of PTH-66.

10 depicts SDS-PAGE Coomassie staining of the final PTH-66 product. The final yield was about 75 mg per 900 mL cell medium (CM). Protein is 3 mg/mL in PTH storage buffer. Proteins were aliquoted and stored at -80°C.

Example 2: Effect of C-terminal truncation of PTH-Fc fusions

PTH-Fc fusion variants containing truncated PTH of various lengths were prepared according to the method outlined in Example 1. The following PTH-Fc fusion variants were generated: PTH-66 (PTH(1-74)-Fc), PTH-67 (PTH(1-64)-Fc), PTH-68 (PTH(1-54)-Fc) ), PTH-69 (PTH(1-44)-Fc), and PTH(1-34)-Fc.

Serum concentrations of PTH after a single subcutaneous treatment of Sprague-Dawley rats using a truncated variant and full-length PTH(1-84)-Fc at 0.25 mg/kg were measured using Immunotopics in vivo. Activity was evaluated using an ELISA kit (60-3000), and the results are shown in FIG. 1 .

As shown in FIG. 1 , C-terminal truncation of PTH surprisingly and unexpectedly improves the exposure and pharmacokinetic properties of PTH-Fc fusions.

As demonstrated in the figure, the truncated PTH-Fc fusion variants of the invention are native-length PTH(1-84)-Fc fusions and teriparatide-Fc (PTH(1-34)-Fc fusions). ) outperform both. Even more surprisingly, PTH(1-74) has a particularly long half-life in vivo when compared to native-length PTH(1-84)-Fc and other analogs.

Example 3: PTH-66 in normal mice

PTH-66 was administered to normal mice at a dose of 50 nmol/kg. Samples were taken at 2, 8, 24, 48, and 72 hours post-dose, and calcium concentrations were plotted and compared to phosphate buffer (vehicle) controls.

FIG. 2 depicts serum calcium levels ( FIG. 2A ) and urine calcium levels ( FIG. 2B ) as a function of time after dosing for PTH-66 (50 nmol/kg dose) in mice. As shown in FIG. 2A , PTH-66 induces a long-lasting increase in serum calcium when compared to PTH (1-84) and vehicle control. Figure 2b, PTH-66 proposes the maintenance of a longer urine calcium levels than PTH (1-84). PTH(1-84) treatment results in a spike in urine calcium within the first few hours, consistent with elevated serum calcium and transient exposure of this molecule. In contrast, despite elevated serum calcium levels, PTH-66 appears to control urine calcium levels over a longer period of time, while maintaining vehicle-like levels for 12-24 hours. In summary, PTH-66 treatment induces a lasting effect on calcium levels.

Example 4: PTH-66 in TxPTx Rats

PTH-66 was administered to normal (intact) and thyroid-parathyroidectomy (TxPTx) rats at various doses of 5, 20, and 60 nmol/kg. Serial samples were taken at 1, 6, 12, 18, 24, 36, 48, and 72 hours post-dose and compared to vehicle controls.

3A and B , TxPTx rats treated with PTH-66 exhibit a dose-dependent PK/PD response with dose-dependent serum Ca++ levels that is elevated and maintained for up to 72 hours post-dose. Figure 3b is a 5 nmol / kg TxPTx rats received the PTH-66, on the other hand presenting the normalized serum calcium levels return to that of an intact rat, as compared with intact rats received the placebo vehicle 20 and 60 nmol / kg PTH- It is suggested that treatment with 66 resulted in higher serum calcium levels 12-48 hours post-dose.

4(ab) presents data from a single dose PK ( FIG. 4A ) and PD ( FIG. 4B ) study from TPTx rats comparing PTH-66 to PTH(1-84). The rat TPTx data showed that 5 nmol/kg of PTH-66 was increased in serum calcium when compared to PTH (1-84), which showed an apparent PK prolongation beyond PTH (1-84), and only a very transient increase in serum calcium. can be returned to normal levels.

In a separate multiple-dose study, PTH-66 was administered subcutaneously daily for 10 days to normal (intact) and thyroid-parathyroidectomy (TxPTx) rats at doses of 2 and 4 nmol/kg. Samples were taken once daily prior to dosing and compared to vehicle control.

5( a d ) presents data from a multi-dose PK ( FIG. 5A ) and PD ( FIGS. 5B-D ) study from daily subcutaneous administration of PTH-66 to TPTx rats. Figure 5a PK data PTH-66 presents the achieved close to the steady state level, the capacity linear PK reaction. As shown in FIG. 5B , 2 and 4 nmol/kg PTH-66 restores and maintains serum calcium to normal range (intact) levels. Figure 5c is to administration of PTH-66 every day also proposes that sikindaneun normalize the serum phosphate levels. Figure 5d shows that while PTH-66 maintains normal serum calcium levels as shown in Figure 5a , the molecule also maintains normal urine calcium levels. These data suggest that daily subcutaneous administration of PTH-66 completely normalizes serum calcium and phosphate, and importantly urine calcium.

Example 5: PTH-66 in primates

PTH-66 was administered to wild-type cynomolgus monkeys as a single dose at a dose of 2 mmol/kg, while other monkeys received a single dose of 5 mmol/kg of natpara (PTH(1-84)). Serial samples were taken at 0, 1, 3, 6, 9 12, 18 24, 36, 48, and 72 hours post-dose and compared to natpara as well as vehicle controls.

Figure 6 (ab) proposes a single dose study data PK (Fig. 6a) and PD (Fig. 6b) from the wild type Sino mole Goose monkeys comparing the PTH-66, and PTH (1-84).

The data in FIG. 6 show that PTH-66 evidently prolongs PK beyond natpara, and that PTH-66 at 2 nmol/kg can stimulate and maintain serum calcium levels beyond that obtained with PTH(1-84). present. Compared to PTH-66, natpara has a shorter lifespan and a more transient effect on serum calcium.

In a separate multiple-dose study, PTH-66 was administered subcutaneously daily for 9 days to wild-type cynomolgus monkeys at a dose of 1 nmol/kg. Samples were taken daily before dosing and 24, 48, 72, and 120 hr after the last dosing and compared to vehicle controls.

7 presents data from a multiple-dose PK and PD study from daily subcutaneous treatment of wild-type cynomolgus monkeys with 1 nmol/kg PTH-66. The PK data show that PTH-66 achieved a relatively flat PK profile, resulting in increased serum calcium and decreased serum phosphate, when compared to vehicle control during the study period.

Example 6: PTH cAMP potency assay

Table 1 below summarizes the results _{of the cAMP potency (EC 50} ) assay for exemplary PTH variants according to the present invention. Table 2 below summarizes the relevant PK data for PTH variants.

Table 1: Selected PTH - Fc in the variant for EC ₅₀ data

Table 2: Selected PTH - Fc in the variant about PK data

As shown in Tables 1 and 2 , all PTH-Fc samples were _{active with sub-nanomolar EC 50} , and had significantly improved PK properties compared to the values of PTH1-84.

Example 6: Multimodal Chromatography in PTH-66 Purification

Protein A chromatography as an initial capture step was performed according to the downstream platform shown in FIG . 11 . After maintaining the elution at low pH 3.5 for 1 hour for virus inactivation, it is neutralized to pH 6 and frozen at -80°C. Most of the impurities except clipped species and host cell proteins were sufficiently removed. The polishing steps described below have been developed to remove residual host cell and process impurities.

Multimodal (also known as mixed mode) chromatographic applications are rapidly growing in antibody purification in that the media can separate compounds with high efficiency through various interactions. Two multimodal column steps are being considered in downstream polishing development, the cationic Capto™ MMC ImpRes and the anionic Capto™ Adhere ImpRes. However, given the complex medium-specific interactions, the purification process is determined by the buffer components, ionic strength, pH, and salt type. Since optimal binding and elution conditions are difficult to estimate, the separation conditions for the two resins were determined through the steps described below to achieve optimal process performance.

PTH-66 (PTH(1-74)-Fc fusion) showed moderate fragmentation from the bioreactor, which, despite increased circulating half-life, may negatively affect its efficacy. The addition of a protease inhibitor to the harvested cell culture prior to downstream processing has been shown to protect against subsequent product degradation. Fragmentation of recombinant proteins in cell culture can be detrimental to drug safety and efficacy. In downstream process development, hydrophobic or charge-altered fragment isoforms generated from the upstream process were successfully isolated from intact Fc-fusion proteins using multimodal chromatography. A flow chart of a production process that meets a fragmentation target of less than 10% is shown in FIG. 12 .

A wide range of parameters including salt concentration and pH were evaluated by performing exhaustive high-throughput screening (HTS) for initial operating conditions of Capto MMC and Adhere ImpRes using 6 μL PreDictor 96-well plates ( FIG. 13A ). and 13b ).

The separation conditions of cation exchange (cationic) Capto MMC ImpRes and anion exchange (anionic) Capto Adhere ImpRes determined using a high-throughput process are shown in FIGS. 14A and 14B . The experimental conditions screened for Capto MMC ImpRes included acetate, MES, and phosphate buffer systems, pH 5.0-7.0 and salt (NaCl) concentrations of 0-750 mM, 50 g/L resin load, and an incubation time of 1 hour. The experimental conditions screened for Capto Adhere ImpRes included acetate, MES, phosphate and Tris buffer systems, pH 5.0 to 8.0 and salt (NaCl) concentrations 0 to 1500 mM, 50 g/L resin load, and an incubation time of 1 hour. did.

It was confirmed that the optimal binding conditions for Capto MMC ImpRes, a cationic resin having the structure shown in FIG. 14c , included 50 mM MES at pH 6.0 in the absence of salt. The optimal flow-through conditions for Capto Adhere ImpRes, an anionic resin having the structure shown in FIG . 14D , were found to include 50 mM acetate buffer at pH 5.0 in the absence of salts.

The second step is to optimize the process at bench-scale, accelerated by the HTS static binding capacity results. Capto MMC ImpRes were run in bind-and-elute mode to maximize impurity clearance capacity. Washing steps were investigated to reduce excessive process- and product-related impurities without unnecessary product loss ( FIG. 15 ). A subsequent Capto Adhere ImpRes step was developed as a fast flow-through (FT) step that allowed the process to meet product quality standards ( FIG. 16 ). MMC and Adhere bench-top validation experiments were performed to demonstrate process robustness with respect to impurity clearance and step yield. PTH-Fc quantification relies on UV280 tracking yield for MMC and Adhere.

17-19 , conditions from the high-throughput study were successfully converted to bench scale. A Capto MMC ImpRes gradient elution tray is shown in Figure 14a . Better resolution was achieved when using a 0.66 x 10 cm (3.4 mL) column with 50 mM MES, pH 6.0, gradient elution 0 to 1 M NaCl. MMC effectively provides HCP, DNA, HMW, and clipped species removal.

Table 3: capto MMC ImpRes gradient dissolution

MMC washing steps were investigated to reduce other process- and product-related impurities. MMC wash optimizations are presented in Table 4 below. The optimal washing conditions were found to be 6 CV of 200 mM NaCl and 3 CV of 250 mM NaCl.

Table 4: MMC cleaning optimization

The Capto Adhere ImpRes flow-through shown in FIG . 17B and presented in Table 5 below is shown to provide additional HCP and HMW removal capabilities. Better resolution was achieved when using a 0.66 x 10 cm (3.4 mL) column with 50 mM sodium acetate buffer flow-through at pH 5.0.

Table 5: capto here ImpRes flow-through

The downstream polishing step design is shown in FIG. 18A . The process robustness with a stable pool based on impurity clearance and process yield is presented in Table 6 below as well as FIG. 15B.

Table 6: Process robustness

The pilot-scale production quality attributes are characterized in FIGS. 19A-F . As shown in these figures, the pilot plant operation (PPO), which is a large scale refinery, has a step yield similar to the yield from the small experimental scale, process development (PD)). By comparing the two yields, it can be concluded that the purification process can be successfully scaled up using the parameters presented above.

MMC eluate and Adhere FT were maintained at ambient temperature and 4-8° C. for 5 and 4 days, respectively, and then analyzed by SEC-HPLC and intact mass spectrometry to determine the stability of in-process samples.

Thus, the Fc-fusion protein fragmentation problem was successfully addressed by using multimodal chromatography and a step elution from a linear gradient was established that achieved a relatively high recovery without compromising robust impurity clearance.

Since the above-described subject matter can be modified in various ways without departing from the scope and spirit of the present invention, all subject matter included in the above description or defined in the appended claims should be construed as explaining and exemplifying the present invention. it is intended to Many variations and modifications may be made to the present invention in light of the above teachings. Accordingly, this description is intended to embrace all such alternatives, variations, and modifications as fall within the scope of the appended claims.

All patents, applications, publications, test methods, literature, and other materials cited herein are hereby incorporated by reference in their entirety as if physically present herein.

SEQUENCE LISTING <110> SHIRE-NPS PHARMACEUTICALS, INC. <120> PARATHYROID HORMONE VARIANTS <130> 250501.000165 <140> PCT/US2020/015634 <141> 2020-01-29 <150> 62/884,730 <151> 2019-08-09 <150> 62/800,744 <151> 2019-02-04 <150> 62/798,113 <151> 2019-01-29 <160> 13 <170> PatentIn version 3.5 <210> 1 <211> 84 <212> PRT <213> Homo sapiens <400> 1 Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn 1 5 10 15 Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30 Asn Phe Val Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser 35 40 45 Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu 50 55 60 Lys Ser Leu Gly Glu Ala Asp Lys Ala Asp Val Asn Val Leu Thr Lys 65 70 75 80 Ala Lys Ser Gln <210> 2 <211> 226 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 2 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 1 5 10 15 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 20 25 30 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 35 40 45 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 50 55 60 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 65 70 75 80 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 85 90 95 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 100 105 110 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 115 120 125 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 130 135 140 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 145 150 155 160 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 165 170 175 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 180 185 190 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 195 200 205 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 210 215 220 Pro Gly 225 <210> 3 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 3 Met Glu Thr Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Leu Trp Leu Pro 1 5 10 15 Asp Thr Thr Gly 20 <210> 4 <211> 19 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 4 Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly 1 5 10 15 Val Gln Cys <210> 5 <211> 16 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 5 Met Pro Leu Leu Leu Leu Leu Pro Leu Leu Trp Ala Gly Ala Leu Ala 1 5 10 15 <210> 6 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 6 Glu Asn Leu Tyr Phe Gln Ser His His His His His His 1 5 10 <210> 7 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic peptide <400> 7 Glu Asn Leu Tyr Phe Gln 1 5 <210> 8 <211> 320 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 8 Met Glu Thr Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Leu Trp Leu Pro 1 5 10 15 Asp Thr Thr Gly Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly 20 25 30 Lys His Leu Asn Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu 35 40 45 Gln Asp Val His Asn Ala Ser Ala Leu Gly Ala Pro Leu Ala Pro Arg 50 55 60 Asp Ala Gly Ser Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val 65 70 75 80 Glu Ser His Glu Lys Ser Leu Gly Glu Ala Asp Lys Ala Asp Asp Lys 85 90 95 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro 100 105 110 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 115 120 125 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 130 135 140 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 145 150 155 160 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 165 170 175 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 180 185 190 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 195 200 205 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 210 215 220 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 225 230 235 240 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 245 250 255 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 260 265 270 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 275 280 285 Ser Arg Trp Gln Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu 290 295 300 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 305 310 315 320 <210> 9 <211> 310 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 9 Met Glu Thr Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Leu Trp Leu Pro 1 5 10 15 Asp Thr Thr Gly Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly 20 25 30 Lys His Leu Asn Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu 35 40 45 Gln Asp Val His Asn Ala Ser Ala Leu Gly Ala Pro Leu Ala Pro Arg 50 55 60 Asp Ala Gly Ser Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val 65 70 75 80 Glu Ser His Glu Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 85 90 95 Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 100 105 110 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 115 120 125 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp 130 135 140 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr 145 150 155 160 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 165 170 175 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 180 185 190 Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 195 200 205 Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 210 215 220 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 225 230 235 240 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 245 250 255 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 260 265 270 Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser 275 280 285 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 290 295 300 Leu Ser Leu Ser Pro Gly 305 310 <210> 10 <211> 300 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 10 Met Glu Thr Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Leu Trp Leu Pro 1 5 10 15 Asp Thr Thr Gly Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly 20 25 30 Lys His Leu Asn Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu 35 40 45 Gln Asp Val His Asn Ala Ser Ala Leu Gly Ala Pro Leu Ala Pro Arg 50 55 60 Asp Ala Gly Ser Gln Arg Pro Arg Lys Lys Asp Lys Thr His Thr Cys 65 70 75 80 Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu 85 90 95 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 100 105 110 Val Thr Cys Val Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 115 120 125 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 130 135 140 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu 145 150 155 160 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 165 170 175 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys 180 185 190 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 195 200 205 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 210 215 220 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 225 230 235 240 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 245 250 255 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln 260 265 270 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 275 280 285 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 290 295 300 <210> 11 <211> 290 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 11 Met Glu Thr Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Leu Trp Leu Pro 1 5 10 15 Asp Thr Thr Gly Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly 20 25 30 Lys His Leu Asn Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu 35 40 45 Gln Asp Val His Asn Ala Ser Ala Leu Gly Ala Pro Leu Ala Pro Arg 50 55 60 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 65 70 75 80 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 85 90 95 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His 100 105 110 Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val 115 120 125 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 130 135 140 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 145 150 155 160 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 165 170 175 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 180 185 190 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser 195 200 205 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 210 215 220 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 225 230 235 240 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val 245 250 255 Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met 260 265 270 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 275 280 285 Pro Gly 290 <210> 12 <211> 329 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic polypeptide <400> 12 Met Glu Thr Pro Ala Gln Leu Leu Phe Leu Leu Leu Leu Leu Trp Leu Pro 1 5 10 15 Asp Thr Thr Gly Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly 20 25 30 Lys His Leu Asn Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu 35 40 45 Gln Asp Val His Asn Ala Ser Ala Leu Gly Ala Pro Leu Ala Pro Arg 50 55 60 Asp Ala Gly Ser Gln Arg Pro Arg Lys Lys Glu Asp Asn Val Leu Val 65 70 75 80 Glu Ser His Glu Lys Ser Leu Gly Glu Ala Asp Lys Ala Asp Val Asn 85 90 95 Leu Thr Lys Ala Lys Ser Gln Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu 225 230 235 240 Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly 325 <210> 13 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Synthetic 6xHis tag <400> 13 His His His His His His 1 5

Claims (47)

A parathyroid hormone (PTH)-Fc fusion protein, wherein the PTH-Fc fusion protein comprises a C-terminally mutated and/or truncated PTH or variant thereof chemically linked to an Fc region of a human IgG antibody, or a derivative thereof. wherein the C-terminally mutated and/or truncated PTH or variant thereof is not PTH(1-34). The method of claim 1 , wherein the C-terminally mutated and/or truncated PTH or variant thereof is mutated PTH(1-84), PTH(1-74), PTH(1-64), PTH(1-54). ), and PTH (1-44). The method of claim 1 , wherein the C-terminally mutated and/or truncated PTH or variant thereof is mutated PTH(1-84), PTH(1-83), or PTH(1-82), or PTH(1). -81), or PTH(1-80), or PTH(1-79), or PTH(1-78), or PTH(1-77), or PTH(1-76), or PTH(1-75) ), or PTH(1-74), or PTH(1-73), or PTH(1-72), or PTH(1-71), or PTH(1-70), or PTH(1-69), or PTH(1-68), or PTH(1-67), or PTH(1-66), or PTH(1-65), or PTH(1-64), or PTH(1-63), or PTH (1-62), or PTH(1-61), or PTH(1-60), or PTH(1-59), or PTH(1-58), or PTH(1-57), or PTH(1) -56), or PTH(1-55), or PTH(1-54), or PTH(1-53), or PTH(1-52), or PTH(1-51), or PTH(1-50) ), or PTH(1-49), or PTH(1-48), or PTH(1-47), or PTH(1-46), or PTH(1-45), or PTH(1-44), or PTH(1-43), or PTH(1-42), or PTH(1-41), or PTH(1-40), or PTH(1-39), or PTH(1-38), or PTH (1-37), or PTH (1-36), or PTH (1-35), or PTH (1-33). The PTH-Fc fusion protein according to any one of claims 1 to 3, wherein the C-terminally mutated and/or truncated PTH or variant thereof is PTH(1-74). 5. The PTH-Fc fusion protein according to any one of claims 1 to 4, wherein the Fc region or derivative thereof is hFcLALA comprising L234A and L235A substitutions. 6. The PTH-Fc fusion protein according to any one of claims 1 to 5, wherein the PTH or variant thereof comprises a F34A substitution, a F34D substitution, a V35S substitution, or a V35T substitution, or a combination thereof. 7. The PTH-Fc fusion protein according to any one of claims 1 to 6, wherein the PTH or variant thereof is glycosylated. 8. The PTH-Fc fusion protein according to any one of claims 1 to 7, wherein the PTH-Fc fusion protein is PTH-66 having the amino acid sequence of SEQ ID NO:8. 8. The PTH-Fc fusion protein according to any one of claims 1 to 7, wherein the PTH-Fc fusion protein is PTH-67 having the amino acid sequence of SEQ ID NO:9. 8. The PTH-Fc fusion protein according to any one of claims 1 to 7, wherein the PTH-Fc fusion protein is PTH-68 having the amino acid sequence of SEQ ID NO:10. 8. The PTH-Fc fusion protein according to any one of claims 1 to 7, wherein the PTH-Fc fusion protein is PTH-69 having the amino acid sequence of SEQ ID NO:11. A nucleic acid encoding the PTH-Fc fusion protein of any one of claims 1-11. An expression vector comprising the nucleic acid of claim 12 . A host cell comprising the nucleic acid of claim 12 or the expression vector of claim 13 . 15. The host cell of claim 14, wherein the host cell is a prokaryotic cell, a yeast cell, an insect cell, or a mammalian cell. 16. The host cell of claim 14 or 15, wherein the host cell is a CHO cell. 12. The PTH-Fc fusion protein according to any one of claims 1 to 11, wherein the serum half-life of the PTH-Fc fusion protein is greater than the serum half-life of PTH(1-84). 12. The PTH-Fc fusion protein according to any one of claims 1 to 11, wherein the serum half-life of the PTH-Fc fusion protein is at least 2-fold greater than the serum half-life of PTH(1-84). 12. The PTH-Fc fusion protein according to any one of claims 1 to 11, wherein the serum half-life of the PTH-Fc fusion protein is at least 10-fold greater than the serum half-life of PTH(1-84). 12. The PTH-Fc fusion protein according to any one of claims 1 to 11, wherein the serum half-life of the PTH-Fc fusion protein is at least 20 times greater than the serum half-life of PTH(1-84). 21. A pharmaceutical composition comprising the PTH-Fc fusion protein of any one of claims 1-11 and 17-20 and a pharmaceutically acceptable carrier. 22. A pharmaceutical dosage form comprising the PTH-Fc fusion protein of any one of claims 1-11 and 17-20, or the pharmaceutical composition of claim 21. 23. The pharmaceutical dosage form of claim 22, wherein the dosage form is a liquid dosage form suitable for administration by injection or infusion. 24. The PTH-Fc fusion protein of any one of claims 1-11 and 17-20, the pharmaceutical composition of claim 21, or the method of claim 22 or 23 to a subject in need of control of serum calcium levels. A method of controlling serum calcium levels in a subject comprising administering an effective amount of a pharmaceutical dosage form. 25. The method of claim 24, wherein the subject has hypoparathyroidism. 26. The method of claim 24 or 25, wherein the PTH-Fc fusion protein, pharmaceutical composition, or pharmaceutical dosage form is administered subcutaneously. 27. The method of any one of claims 24-26, wherein the PTH-Fc fusion protein, pharmaceutical composition, or pharmaceutical dosage form is administered once daily. 28. The method of any one of claims 24-27, wherein the PTH-Fc fusion protein, pharmaceutical composition, or pharmaceutical dosage form is administered once a week. 29. The method of any one of claims 24-28, wherein the PTH-Fc fusion protein, pharmaceutical composition, or pharmaceutical dosage form is administered to the subject in an amount of from about 20 μg/day to about 100 μg/day of the PTH-Fc fusion protein. how it is provided. 24. Use of the PTH-Fc fusion protein of any one of claims 1-11 and 17-20, the pharmaceutical composition of claim 21, or the pharmaceutical dosage form of claim 22 or 23 to a subject having hypoparathyroidism. A method of treating a subject comprising administering an effective amount. 31. The method of claim 30, wherein the PTH-Fc fusion protein, pharmaceutical composition, or pharmaceutical dosage form is administered subcutaneously. 32. The method of claim 30 or 31, wherein the PTH-Fc fusion protein, pharmaceutical composition, or pharmaceutical dosage form is administered once daily. 33. The method of any one of claims 30-32, wherein the PTH-Fc fusion protein, pharmaceutical composition, or pharmaceutical dosage form is administered to the subject in an amount of from about 20 μg/day to about 100 μg/day of the PTH-Fc fusion protein. how it is provided. 23. The PTH-Fc fusion protein of any one of claims 1-11 and 17-20, the pharmaceutical composition of claim 21, or the pharmaceutical composition of claim 21, or claim 22 or 23 to a subject having hypocalcemia due to hypoparathyroidism. A method of treating a subject comprising administering an effective amount of the pharmaceutical dosage form of claim 23 . 35. The method of claim 34, wherein the PTH-Fc fusion protein, pharmaceutical composition, or pharmaceutical dosage form is administered subcutaneously. 36. The method of claim 34 or 35, wherein the PTH-Fc fusion protein, pharmaceutical composition, or pharmaceutical dosage form is administered once daily. 37. The method of any one of claims 34-36, wherein the PTH-Fc fusion protein, pharmaceutical composition, or pharmaceutical dosage form is administered to the subject in an amount from about 20 μg/day to about 100 μg/day of the PTH-Fc fusion protein. how it is provided. 24. The PTH-Fc fusion protein according to any one of claims 1 to 11 and 17 to 23, wherein the PTH-Fc fusion protein comprises one copy of PTH and one copy of Fc; A pharmaceutical composition, or pharmaceutical dosage form. 24. The PTH-Fc fusion protein according to any one of claims 1 to 11 and 17 to 23, wherein the PTH-Fc fusion protein comprises two copies of PTH and one copy of Fc; A pharmaceutical composition, or pharmaceutical dosage form. C-terminally truncated (PTH)-Fc fusion protein comprising PTH(1-74) or a variant thereof comprising purifying the parathyroid hormone (PTH)-Fc fusion protein using a multimodal chromatography resin How to manufacture. 41. The method of claim 40, wherein the multimodal chromatography resin comprises a cation exchange resin and an anion exchange resin. 42. The method of claim 41, wherein the cation exchange resin is Capto MMC ImpRes. 43. The method of claim 42, wherein the (PTH)-Fc fusion protein is bound at pH 6.0 in 50 mM MES buffer. 42. The method of claim 41, wherein the anion exchange resin is Capto Adhere ImpRes. 45. The method of claim 44, wherein the (PTH)-Fc fusion protein is eluted at pH 5.0 in 50 mM acetate buffer. 46. The method of any one of claims 40-45, further comprising progressively eluting the (PTH)-Fc fusion protein from the multimodal chromatography resin. 47. The method of any one of claims 40-46, wherein the purified (PTH)-Fc fusion protein comprises less than 10% total fragmentation impurities.

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